Carbofluorescein Lactone Ion Indicators and Their Applications

ABSTRACT

Fluorescent dyes useful for preparing fluorescent metal ion indicators, the fluorescent indicators themselves, and the use of the fluorescent indicators for the detection, discrimination and quantification of metal cations are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 13/374,967, filed Jan. 25, 2012, which is acontinuation-in-part of U.S. patent application Ser. No. 12/932,683,filed Mar. 2, 2011, which is a divisional of U.S. patent applicationSer. No. 12/040,753, filed Feb. 29, 2008, which claims priority under 35U.S.C. §119(e) to U.S. provisional patent application Ser. No.60/923,452, filed Apr. 13, 2007, each of which is hereby incorporated byreference.

BACKGROUND

Metal ions play important roles in many biological systems. Cellsutilize metal ions for a wide variety of functions, such as regulatingenzyme activities, protein structures, cellular signaling, as catalysts,as templates for polymer formation and as regulatory elements for genetranscription. Metal ions can also have a deleterious effect whenpresent in excess of bodily requirements or capacity to excrete. A largenumber of natural and synthetic materials are known to selectively ornon-selectively bind to or chelate metal ions. Ion chelators arecommonly used in solution for in vivo control of ionic concentrationsand detoxification of excess metals, and as in vitro buffers. Ionchelators can be used as optical indicators of ions when bound to afluorophore, and may be useful in the analysis of cellularmicroenvironments or dynamic properties of proteins, membranes andnucleic acids. For example, Ca²⁺ ions play an important role in manybiological events, and so the determination of intracellular Ca²⁺ is animportant biological application.

Fluorescent indicators utilizing a BAPTA chelator have beenpredominantly used for intracellular calcium detections (see U.S. Pat.No. 4,603,209; U.S. Pat. No. 5,049,673; U.S. Pat. No. 4,849,362; U.S.Pat. No. 5,453,517; U.S. Pat. No. 5,501,980; U.S. Pat. No. 5,459,276;U.S. Pat. No. 5,501,980; U.S. Pat. No. 5,459,276; and U.S. Pat. No.5,516,911; each of which is hereby incorporated by reference).Fluorescein-based fluorescent calcium indicators (such as Fluo-3 andFluo-4) are the most common fluorescent indicators used in biologicalassays. However, these existing xanthene-based calcium indicatorstypically have either small cell-induced excitation and fluorescencewavelength change, and/or low calcium-induced fluorescence enhancement,resulting in low detection sensitivity and high assay background. Inaddition, the existing xanthene-based calcium indicators have shortemission wavelength, which often interferes with the fluorescence ofsome agonists and/or antagonists to be screened. Another drawback isresulted from the spontaneous hydrolysis of monoalkylatedfluorescein-based indicators in cell medium, generating significantassay background.

In view of the existing drawbacks for currently used fluorescein-basedfluorescent calcium indicators, what is needed are improved compositionsand methods that offer sensitive detection of small variations incalcium and other ion concentrations, with a rapid response and a strongfluorescence signal. Also needed are fluorescent indicators that can bereadily loaded into live cells with large spectral shift andfluorescence enhancement. Another preferable property is that theindicators have emission at longer wavelength to reduce the interferencefrom the fluorescence of agonists and antagonist that often havefluorescence at short wavelength. In addition, compositions and methodsthat are less susceptible to the effects of external changes (such astemperature) are preferred for high throughput screening and highcontent analysis.

The present application is directed to a family of fluorescent dyes thatare useful for preparing fluorescent metal ion indicators. Theindicators include a carbofluorescein lactone fluorophore that isincorporated with an ionophore, and are useful for the detection,discrimination and quantification of metal cations. The fluorescentindicators of this invention demonstrate unexpected larger spectralshift upon cell-induced hydrolysis, unexpected emission shift to thelonger wavelength, better stability in the presence of cells and bettercellular retention compared to the existing fluorescein ion indicators.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. The chemical structures of Fluo-3, Fluo-3 AM, Fluo-4 and Fluo-4AM.

FIG. 2. The spontaneous hydrolysis of Fluo-3 AM and Compound 128.

FIG. 3. Calcium responses of Compound 85 in CHO-K1 cells measured with afluorescence microplate reader. CHO-K1 cells are seeded overnight at50,000 cells per 100 μl per well in a 96-well black wall/clear bottomcostar plate. The growth medium is removed, and the cells are incubatedwith 100 μl of Compound 85 at 5 μM in Hanks and Hepes buffer in thepresence of 1 mM probenecid for 3 hours at 37° C., 5% CO₂ incubator. ATP(50 μL/well) was added by FlexStation to achieve the final desiredconcentrations.

FIG. 4. Calcium responses of Compound 125 in CHO-K1 cells measured witha fluorescence microplate reader. CHO-K1 cells are seeded overnight at50,000 cells per 100 μl per well in a 96-well black wall/clear bottomcostar plate. The growth medium is removed, and the cells are incubatedwith 100 μl of Compound 125 at 5 μM in Hanks and Hepes buffer in thepresence of 1 mM probenecid for 3 hours at 37° C., 5% CO₂ incubator. ATP(50 μL/well) was added by FlexStation to achieve the final desiredconcentrations.

FIG. 5. Calcium responses of Compound 79 (5 μM) measured in fluorescenceexcitation in PBS buffer solution. The calcium concentrations areindicated in the graph in the unit of μM.

FIG. 6. Calcium responses of Compound 79 (5 μM) measured in fluorescenceemission in PBS buffer solution. The calcium concentrations areindicated in the graph in the unit of μM.

FIG. 7. The cell-induced absorption spectral shift comparison of Fluo-3AM and Compound 16. The absorption spectra are normalized at theirmaximum absorption peak. Solid lines represent the absorption spectra ofCompound 16 in the absence of CHO cells (left) and in the presence ofCHO cells (right) respectively. Dot lines represent the absorptionspectra of Fluo-3 AM in the absence of CHO cells (left) and in thepresence of CHO cells (right) respectively.

FIG. 8. The cell-induced absorption spectral shift comparison of Fluo-4AM and Compound 85. The absorption spectra are normalized at theirmaximum absorption peak. Solid lines represent the absorption spectra ofCompound 85 in the absence of CHO cells (left) and in the presence ofCHO cells (right) respectively. Dot lines represent the absorptionspectra of Fluo-4 AM in the absence of CHO cells (left) and in thepresence of CHO cells (right) respectively.

FIG. 9. The esterase-induced hydrolysis comparison of Compound 85 andFluo-4 AM in live cells. Both of the compounds generate the samecalcium-binding BAPTA moiety (marked in the rectangle). However, in livecells Compound 85 generates a fluorophore that carries an additionalcarboxy group, which significantly enhances the indicator fluorescenceand retains the indicator from leaking out of cells.

FIG. 10. Calcium responses of Compound 85 in CHO-K1 cells measured witha fluorescence microscope. CHO-K1 cells are seeded overnight at 50,000cells per 100 μl per well in a 96-well black wall/clear bottom costarplate. The growth medium is removed, and the cells are incubated with100 μl of Compound 85 at 5 μM in Hanks and Hepes buffer in the presenceof 1 mM probenecid for 3 hours at 37° C., 5% CO₂ incubator. ATP (3 μM,50 μL/well) was added, and imaged with a fluorescence microscope usingTRITC channel.

FIG. 11. Calcium responses of Compound 125 in CHO-K1 cells measured witha fluorescence microscope. CHO-K1 cells are seeded overnight at 50,000cells per 100 μl per well in a 96-well black wall/clear bottom costarplate. The growth medium is removed, and the cells are incubated with100 μl of Compound 125 at 5 μM in Hanks and Hepes buffer in the presenceof 1 mM probenecid for 3 hours at 37° C., 5% CO₂ incubator. ATP (3 μM,50 μL/well) was added, and imaged with a fluorescence microscope usingTRITC channel.

DEFINITIONS

The following definitions are set forth to illustrate and define themeaning and scope of the various terms used to describe the inventionherein.

The term “organic substituent”, as used herein, refers to acarbon-containing organic radical that incorporates straight, branchedchain or cyclic radicals having up to 50 carbons, unless the chainlength or ring size is limited thereto. The organic substituent mayinclude one or more elements of unsaturation, such as carbon-carbondouble or triple bonds. Organic substituents may include alkyl,alkylene, alkenyl, alkenylene and alkynyl moieties, among others.

The term “alkyl,” as used herein, by itself or as part of another group,refers to straight, branched chain or cyclic radicals having up to 50carbons, unless the chain length or ring size is limited thereto, suchas methyl, ethyl, propyl, cyclopropanyl, isopropyl, butyl, t-butyl,isobutyl, pentyl, hexyl, cyclohexyl, isohexyl, heptyl,4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, and decyl,among others.

The term “alkylene,” as employed herein, by itself or as part of anothergroup, refers to straight, branched chain or cyclic divalent radicalshaving up to 50 carbons, unless the chain length or ring size is limitedthereto. Typical examples include methylene (—CH₂—), ethylene(—CH₂CH₂—), propylene, butylene, pentylene, hexylene, heptylene,octylene, nonylene, and decylene, among others.

The term “alkenyl,” as used herein, by itself or as part of anothergroup, means a straight, branched chain or cyclic radical having 2-50carbon atoms and one or more carbon-carbon double bonds, unless thechain length or ring size is limited thereto, such as ethenyl,1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, and 2-butenyl,among others. The alkenyl chain may be 2 to 10 carbon atoms in length.Alternatively, the alkenyl chain may be 2 to 4 carbon atoms in length.

The term “alkenylene,” as used herein, by itself or as part of anothergroup, means straight, branched chain or cyclic divalent radical having2-50 carbon atoms, unless the chain length or ring size is limitedthereto, said straight, branched chain or cyclic radical containing atleast one carbon-carbon double bond. Typical examples include ethenylene(—CH═CH—), propenylene (—CH═CHCH₂— and —CH₂CH═CH—), n-butenylene, and3-methyl-2-pentenylene, hexenylene, heptenylene, octenylene, nonenylene,and decenylene, among others.

The term “alkynyl,” as used herein, by itself or as part of anothergroup, means a straight, branched chain or cyclic radical of 2-50 carbonatoms, unless the chain length or ring size is limited thereto, havingat least one carbon-carbon triple bond between two of the carbon atomsin the chain, such as acetylenyl, 1-propynyl, and 2-propynyl, amongothers. The alkynyl chain may be 2 to 10 carbon atoms in length.Alternatively, the alkynyl chain may be from 2 to 4 carbon atoms inlength.

The term “alkynylene” as used herein, by itself or as part of anothergroup, means a straight, branched chain or cyclic divalent radicalhaving 2-50 carbon atoms, unless the chain length or ring size islimited thereto, that contains at least one carbon-carbon triple bond.Typical examples include ethynylene (—C≡C—), propynylene (—C≡CCH₂— and—CH₂C≡C—), n-butynylene, 4-methyl-2-pentynylene, 1-butynylene,2-butynylene, 3-butynylene, 4-butynylene, pentynylene, hexynylene,heptynylene, octynylene, nonynylene, and decynylene, among others.

The term “alkoxy” as used herein, by itself or as part of another group,refers to any of the above radicals linked via an oxygen atom. Typicalexamples include methoxy, ethoxy, isopropyloxy, sec-butyloxy,n-butyloxy, t-butyloxy, n-pentyloxy, 2-methylbutyloxy, 3-methylbutyloxy,n-hexyloxy, and 2-ethylbutyloxy, among others. Alkoxy also may includePEG groups (—OCH₂CH₂O—) or alkyl moieties that contain more than oneoxygen atom.

The term “aryl,” as employed herein, by itself or as part of anothergroup, refers to an aryl or aromatic ring system containing 1 to 4unsaturated rings (each ring containing 6 conjugated carbon atoms and noheteroatoms) that are optionally fused to each other or bonded to eachother by carbon-carbon single bonds, that is optionally furthersubstituted as described below. Examples of aryl ring systems include,but are not limited to, substituted or unsubstituted derivatives ofphenyl, biphenyl, o-, m-, or p-terphenyl, 1-naphthyl, 2-naphthyl, 1-,2-, or 9-anthryl, 1-, 2-, 3-, 4-, or 9-phenanthrenyl and 1-, 2- or4-pyrenyl. Aryl substituents may include phenyl, substituted phenyl,naphthyl or substituted naphthyl.

The term “heteroaryl,” as employed herein, by itself or as part ofanother group, refers to groups having 5 to 14 ring atoms; 6, 10 or 14 πelectrons shared in a cyclic array; and containing carbon atoms and 1,2, 3, or 4 oxygen, nitrogen or sulfur heteroatoms (where examples ofheteroaryl groups are: thienyl, benzo[b]thienyl, naphtho[2,3-b]thienyl,thianthrenyl, furyl, pyranyl, isobenzofuranyl, benzoxazolyl, chromenyl,xanthenyl, phenoxathiinyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl,pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl,3H-indolyl, indolyl, indazolyl, purinyl, 4H-quinolizinyl, isoquinolyl,quinolyl, phthalazinyl, naphthyridinyl, quinazolinyl, cinnolinyl,pteridinyl, carbazolyl, phenanthridinyl, acridinyl, perimidinyl,phenanthrolinyl, phenazinyl, isothiazolyl, phenothiazinyl, isoxazolyl,furazanyl, phenoxazinyl, and tetrazolyl groups).

Any aryl or heteroaryl ring system is unsubstituted or optionally andindependently substituted by any synthetically accessible and chemicallystable combination of substituents, such as H, halogen, cyano, sulfo,alkali or ammonium salt of sulfo, nitro, carboxy, alkyl, perfluoroalkyl,alkoxy, alkylthio, amino, monoalkylamino, dialkylamino or alkylamido,the alkyl portions of which having 18 or fewer carbons.

The terms “halogen” or “halo” as employed herein, by itself or as partof another group, refers to chlorine, bromine, fluorine or iodine.

The terms “AM ester” or “AM” as employed herein, by itself or as part ofanother group, refers to an acetoxymethyl ester of a carboxylic acid ora phenol.

The terms “amino” or “amine” include NH₂, “monoalkylamine” or“monoalkylamino,” and “dialkylamine” or “dialkylamino” The terms“monoalkylamine” and “monoalkylamino,” “dialkylamine” and “dialkylaminoas employed herein, by itself or as part of another group, refers to thegroup NH₂ where one hydrogen has been replaced by an alkyl group, asdefined above.

The terms “dialkylamine” and “dialkylamino” as employed herein, byitself or as part of another group, refers to the group NH₂ where bothhydrogens have been replaced by alkyl groups, as defined above.

The term “hydroxyalkyl,” as employed herein, by itself or as part ofanother group, refers to an alkyl group where one or more hydrogensthereof are substituted by one or more hydroxyl moieties.

The term “haloalkyl,” as employed herein, by itself or as part ofanother group, refers to an alkyl group where one or more hydrogensthereof are substituted by one or more halo moieties. Typical examplesinclude chloromethyl, fluoromethyl, difluoromethyl, trifluoromethyl,trichloroethyl, trifluoroethyl, fluoropropyl, and bromobutyl, amongothers.

The term “haloalkenyl,” as employed herein, by itself or as part ofanother group, refers to an alkenyl group where one or more hydrogensthereof are substituted by one or more halo moieties.

The term “haloalkynyl,” as employed herein, by itself or as part ofanother group, refers to an alkynyl group where one or more hydrogensthereof are substituted by one or more halo moieties.

The term “carboxyalkyl,” as employed herein, by itself or as part ofanother group, refers to an alkyl group where one or more hydrogensthereof are substituted by one or more carboxylic acid moieties.

The term “heteroatom” as used herein, by itself or as part of anothergroup, means an oxygen atom (“O”), a sulfur atom (“S”) or a nitrogenatom (“N”). It will be recognized that when the heteroatom is nitrogen,it may form an NR₁R₂ moiety, where R₁ and R₂ are, independently from oneanother, hydrogen or alkyl, or together with the nitrogen to which theyare bound, form a saturated or unsaturated 5-, 6-, or 7-membered ring.

The term “chelator”, “chelate”, “chelating group”, “ionophore”, or“ionophoric moiety” as used herein, by itself or as part of anothergroup, refers to a chemical moiety that binds to, or complexes with, oneor more metal ions, such as lithium, calcium, sodium, magnesium, zinc,potassium, and/or other biologically important metal ions. The bindingaffinity of a chelator for a particular metal ion can be determined bymeasuring the dissociation constant between that chelator and that ion.Chelators may include one or more chemical moieties that bind to, orcomplex with, a cation or anion. Examples of suitable chelators include1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA),bipyridyl (bipy); terpyridyl (terpy); ethylenediaminetetraacetic acid(EDTA); crown ethers; aza-crown ethers; succinic acid; citric acid;salicylic acids; histidines; imidazoles;ethyleneglycol-bis-(beta-aminoethyl ether) N,N′-tetraacetic acid (EGTA);nitroloacetic acid; acetylacetonate (acac); sulfate; dithiocarbamates;carboxylates; alkyldiamines; ethylenediamine (en); diethylenetriamine(dien); nitrate; nitro; nitroso; glyme; diglyme;bis(acetylacetonate)ethylenediamine (acacen);1,4,7,10-tetraazacyclododecanetetraacetic acid (DOTA),1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A),1-oxa-4,7,10-triazacyclododecane-triacetic acid (OTTA),1,4,7-triazacyclononanetriacetic acid (NOTA),1,4,8,11-tetraazacyclotetra-decanetetraacetic acid (TETA),DOTA-N-(2-aminoethyl)amide; DOTA-N-(2-aminophenethyl)amide; and1,4,8,11-tetraazacyclotetradecane, among others.

The term “BAPTA” or “1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraaceticacid” as used herein, by itself or as part of another group, refers tothe following ring structure or its derivatives, such as esters, amides,carbamates and so on:

The term “fluorophore or fluorophore moiety” as used herein, by itselfor as part of another group, means a molecule or a portion of a moleculewhich exhibits fluorescence. By fluorescence is meant that the moleculeor portion of a molecule can absorb excitation energy having a givenwavelength and emit energy at a different wavelength. The intensity andwavelength of the emitted energy depend on the fluorophore, the chemicalenvironment of the fluorophore, and the specific excitation energy used.Exemplary fluorophores include, but are not limited to, fluoresceins,rhodamines, coumarins, oxazines, cyanines, pyrenes, and other polycyclicaromatic molecules.

The term “xanthene”, or “xanthene derivative”, as used herein, by itselfor as part of another group, means any compounds or substituents thatcontain one or more of the following fused ring structures or itsderivatives:

The term “fluorescein” as used herein, by itself or as part of anothergroup, means any compounds or substituents that contain one or more ofthe following fused ring structures or its derivatives:

The term “carbofluorescein” as used herein, by itself or as part ofanother group, means any compounds or substituents that contain one ormore of the following fused ring structures or its derivatives:

The term “carbofluorescein lactone” as used herein, by itself or as partof another group, means any compounds or substituents that contain oneor more of the following fused ring structures or its derivatives:

The term “substituted,” as used herein, refers to the formal replacementof a hydrogen on a chemical moiety or functional group with analternative radical. Where a compound, chemical moiety or functionalgroup is described as substituted, the alternative radical substituentmoiety is generally selected from the group consisting of hydroxy, oxo,nitro, trifluoromethyl, halogen, alkoxy, alkylenedioxy, aminoalkyl,aminoalkoxy, amino, monoalkylamino, dialkylamino, alkylcarbonylamino,alkoxycarbonylamino, alkoxycarbonyl, carboxy, hydroxyalkoxy,alkoxyalkoxy, monoalkylaminoalkoxy,dialkylaminoalkoxymono(carboxyalkyl)amino, bis(carboxy-alkyl)amino,alkoxycarbonyl, alkynylcarbonyl, alkylsulfonyl, alkenylsulfonyl,alkynylsulfonyl, arylsulfonyl, alkylsulfinyl, alkylsulfonamido,arylsulfonamido, carboxyalkoxy, carboxyalkyl, carboxyalkylamino, cyano,trifluoromethoxy, perfluoroethoxy, guanidine, amidino, oxyguanidino,alkylimino, formylimino, acyl nitrile, acyl azide, acetyl azide,dichlorotriazene, isothiocyante, sulfonyl halide, sulfosuccinimidylester, isocyante, acyl halide, aldehyde, haloacetamide, maleimido,aziridinyl, alkylthio (disulfide), acrylo, haloalkylcarbonyl, boronate,hydrazide, semicarbazide, carbohydrazide, arylalkyl, heteroarylalkyl,cycloalkylalkyl, cycloalkenylalkyl, cycloheteroalkylalkyl, andcycloheteroalkenylalkyl.

The term “indicator compound” refers to the compounds of the invention,specifically to those compounds having utility as fluorescent metal ionindicators, as well as their acylated or otherwise protected precursorcompounds, such as the acetoxymethyl ester derivatives suitable foradding to samples containing biological cells.

The term “screening” refers to the testing and/or evaluation of amultiplicity of molecules or compounds for a selected property ortherapeutic utility. Screening is typically a repetitive, or iterativeprocess. A multiplicity of candidate molecules may be screened for theirability to bind to a target molecule which is capable of denaturingand/or unfolding. For example, a multiplicity of candidate molecules maybe evaluated for their ability to bind to a target molecule (e.g., aprotein receptor) in a thermal shift assay. If none of a selected subsetof molecules from the multiplicity of candidate molecules (for example,a combinatorial library) binds to the target molecule, then a differentsubset may be tested for binding in the thermal shift assay.

The term “high-throughput”, as used herein, encompasses screeningactivity in which human intervention is minimized, and automation ismaximized. For example, high-throughput screening may include any of avariety of automated processes, including for example the automation ofpipetting, mixing, and/or heating, the software-controlled generation ofthermal unfolding information, and the software-controlled comparisonsof thermal unfolding information. Alternatively, a high-throughputmethod is one in which hundreds of compounds can be screened per 24 hourperiod by a single individual operating a single suitable apparatus.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present application is directed to fluorescent dyes useful forpreparing fluorescent metal ion indicators, the fluorescent indicatorsthemselves, and the use of the fluorescent indicators for the detection,discrimination and quantification of metal cations.

In one aspect of the invention, the compounds of the invention may bedescribed by Formula 1:

In this embodiment, A is acyl or acyloxymethyl; R¹-R⁷ are independentlyH, alkyl, halogen, carboxy, alkoxy, aryloxy, thiol, alkylthiol,arylthiol, azido, nitro, nitroso, cyano, amino, hydroxy, phosphonyl,sulfonyl, carbonyl, boronic acid, aryl or heteroaryl; or alkyl, oralkoxy that is itself optionally substituted one or more times byhalogen, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid,aryl or heteroaryl; R²⁰ and R²¹ are independently alkyl, carboxyalkyl,aryl or heteroaryl; or alkyl, or alkoxy that is itself optionallysubstituted one or more times by halogen, amino, hydroxy, phosphonyl,sulfonyl, carbonyl, boronic acid, aryl or heteroaryl; Y¹ and Y², Y¹ andY³ or Y² and Y⁴ form a metal ion chelator in combination with C, H, N, Oor S atoms provided that at least one of Y¹Y², and Y³ is NH, N-alkyl orN(CH₂)_(n)COOCH₂OAc wherein n is 1-10.

In another aspect, A is acetyl; R¹-R⁷ are independently H, alkyl,halogen, carboxy, aryl or heteroaryl; R²⁰ and R²¹ are independentlyalkyl, aryl or heteroaryl; Y¹ and Y² or Y¹ and Y³ combine to form acell-permeable metal ion-chelating moiety.

In another aspect, A is acetyl or acetoxymethyl; R¹-R⁷ are independentlyH, alkyl, halogen, carboxy, aryl or heteroaryl; R²⁰ and R²¹ areindependently alkyl, aryl or heteroaryl; Y¹ and Y² or Y¹ and Y³ combineto form a cell-permeable metal ion-chelating crown ether moiety.

In another aspect, A is acetyl; R¹-R⁷ are independently H, alkyl,halogen, aryl or heteroaryl; R²⁰ and R²¹ are independently alkyl, arylor heteroaryl; Y¹ and Y² or Y¹ and Y³ combine to form a cell-permeablemetal ion-chelating azacrown ether moiety.

In another aspect, A is acetyl; R¹-R⁷ are independently H, alkyl,halogen, carboxy, aryl or heteroaryl; R²⁰ and R²¹ are independentlyalkyl, aryl or heteroaryl; Y¹ and Y² or Y¹ and Y³ combine to form acell-permeable metal ion-chelating cryptand moiety.

In another aspect of the invention, the compounds of the invention maybe described by Formula 2:

In this embodiment, substituents R¹-R⁶ and Y⁴ are independently H,alkyl, halogen, carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol,azido, nitro, nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl,carbonyl, boronic acid, aryl or heteroaryl; or alkyl, or alkoxy that isitself optionally substituted one or more times by halogen, amino,hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl orheteroaryl; R²⁰ and R²¹ are independently alkyl, carboxyalkyl, aryl orheteroaryl; or alkyl, or alkoxy that is itself optionally substitutedone or more times by halogen, amino, hydroxy, phosphonyl, sulfonyl,carbonyl, boronic acid, aryl or heteroaryl; X¹, X² and Y², or X¹, X² andY³ combine to form a metal ion-chelating moiety; A is an alkyl having1-10 carbons. In certain embodiments, A is methyl.

In another aspect of the invention, A is methyl; R¹-R⁶ and Y⁴ areindependently H, alkyl, halogen, carboxy, aryl or heteroaryl; R²⁰ andR²¹ are independently alkyl, aryl or heteroaryl; X¹, X² and Y² or X¹, X²and Y³ combine to form a metal ion-chelating moiety.

In another aspect of the invention, A is methyl; R¹-R⁶ and Y⁴ areindependently H, alkyl, fluoro or chloro; R²⁰ and R²¹ are methyl; X¹, X²and Y², or X¹, X² and Y³ combine to form a metal ion-chelating moiety.

In another aspect of the invention, the compounds of the invention maybe described by Formula 3:

In this embodiment, substituents R¹-R⁶ and Y³ are independently H,halogen, carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido,nitro, nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl,boronic acid, aryl or heteroaryl; or alkyl, or alkoxy that is itselfoptionally substituted one or more times by halogen, amino, hydroxy,phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or heteroaryl; R²⁰and R²¹ are independently alkyl, carboxyalkyl, aryl or heteroaryl; oralkyl, or alkoxy that is itself optionally substituted one or more timesby halogen, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronicacid, aryl or heteroaryl; X¹, X² and Y¹ or X¹, X² and Y⁴ combine to forma metal ion-chelating moiety; A is an alkyl having 1-10 carbons. Incertain embodiments, A is methyl.

In another aspect of the invention, A is methyl; R¹-R⁶ and Y³ areindependently H, alkyl, halogen, carboxy, aryl or heteroaryl; R²⁰ andR²¹ are independently alkyl, aryl or heteroaryl; X¹, X² and Y¹ combineto form a metal ion-chelating moiety.

In another aspect of the invention, A is methyl; R¹-R⁶ and Y³ areindependently H, alkyl, fluoro or chloro; R²⁰ and R²¹ are methyl; X¹, X²and Y¹ or X¹, X² and Y⁴ combine to form a metal ion-chelating moiety.

In another aspect of the invention, the compounds of the invention maybe described by Formula 4:

In this embodiment, substituents R¹-R¹² are independently H, halogen,carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro,nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronicacid, aryl or heteroaryl; or alkyl, or alkoxy that is itself optionallysubstituted one or more times by halogen, amino, hydroxy, phosphonyl,sulfonyl, carbonyl, boronic acid, aryl or heteroaryl; R²⁰ and R²¹ areindependently alkyl, carboxyalkyl, aryl or heteroaryl; or alkyl, oralkoxy that is itself optionally substituted one or more times byhalogen, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid,aryl or heteroaryl; R²⁴-R²⁷ are independently hydrogen, alkyl,carboxyalkyl, aryl or heteroaryl; or alkyl, or alkoxy that is itselfoptionally substituted one or more times by halogen, amino, hydroxy,phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or heteroaryl; A isan alkyl having 1-10 carbons. In certain embodiments, A is methyl; Z isan acyloxymethyl having 3-10 carbons.

In another aspect of the invention, A is alkyl; R¹-R¹² are independentlyH, alkyl, aryl or halogen; R²⁰ and R²¹ are independently alkyl, aryl orheteroaryl; R²⁴-R²⁷ are independently hydrogen, alkyl or aryl; Z is anacyloxymethyl having 3-10 carbons.

In another aspect of the invention, A is methyl; R¹-R¹² areindependently H, alkyl, fluoro or chloro; R²⁰ and R²¹ are methyl;R²⁴-R²⁷ are independently hydrogen or alkyl; Z is acetoxymethyl.

In another aspect of the invention, the compounds of the invention maybe described by Formula 5:

In this embodiment, substituents R¹-R¹² are independently H, halogen,carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro,nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronicacid, aryl or heteroaryl; or alkyl, or alkoxy that is itself optionallysubstituted one or more times by halogen, amino, hydroxy, phosphonyl,sulfonyl, carbonyl, boronic acid, aryl or heteroaryl; R²⁰-R²⁷ areindependently alkyl, carboxyalkyl, aryl or heteroaryl; or alkyl, oralkoxy that is itself optionally substituted one or more times byhalogen, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid,aryl or heteroaryl; A is an alkyl having 1-10 carbons. In certainembodiments, A is methyl; Z is an acyloxymethyl having 3-10 carbons.

In another aspect of the invention, A is alkyl; R¹-R¹² are independentlyH, alkyl, aryl or halogen; R²⁰ and R²¹ are independently alkyl, aryl orheteroaryl; R²⁴-R²⁷ are independently hydrogen, alkyl or aryl; Z is anacyloxymethyl having 3-10 carbons.

In another aspect of the invention, A is methyl; R¹-R¹² areindependently H, alkyl, fluoro or chloro; R²⁰ and R²¹ are methyl;R²⁴-R²⁷ are independently hydrogen or alkyl; Z is acetoxymethyl.

In another aspect of the invention, the compounds of the invention maybe described by Formula 6:

In this embodiment, substituents R¹-R¹² are independently H, halogen,carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro,nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronicacid, aryl or heteroaryl; or alkyl, or alkoxy that is itself optionallysubstituted one or more times by halogen, amino, hydroxy, phosphonyl,sulfonyl, carbonyl, boronic acid, aryl or heteroaryl; R²⁰ and R²¹ areindependently alkyl, carboxyalkyl, aryl or heteroaryl; or alkyl, oralkoxy that is itself optionally substituted one or more times byhalogen, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid,aryl or heteroaryl; A is an alkyl having 1-10 carbons. In certainembodiments, A is methyl; Z is an acyloxymethyl having 3-10 carbons. Incertain embodiments, Z is acetoxymethyl.

In another aspect of the invention, A is alkyl; R¹-R¹² are independentlyH, alkyl, aryl or halogen; R²⁰ and R²¹ are independently alkyl, aryl orheteroaryl; A is methyl; Z is acetoxymethyl.

In another aspect of the invention, A is methyl; R¹-R¹² areindependently H, alkyl, fluoro or chloro; R²⁰ and R²¹ are methyl; Z isacetoxymethyl.

In some embodiments, the compound is described by Formula 17:

wherein R¹-R¹² are independently H, halogen, carboxy, alkoxy, aryloxy,thiol, alkylthiol, arylthiol, azido, nitro, nitroso, cyano, amino,hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl orheteroaryl; or alkyl, or alkoxy that is itself optionally substitutedone or more times by halogen, amino, hydroxy, phosphonyl, sulfonyl,carbonyl, boronic acid, aryl or heteroaryl; R²⁰ and R²¹ areindependently alkyl, carboxyalkyl, aryl or heteroaryl; or alkyl, oralkoxy that is itself optionally substituted one or more times byhalogen, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid,aryl or heteroaryl; A is an acyl or an acyloxymethyl having 1-10carbons; and Z is an acyloxymethyl having 3-10 carbons.

In some embodiments of Formula 17, A is acetyl or acetoxymethyl. Incertain embodiments of Formula 17, A is acetyl and Z is an acyloxymethylhaving 3-10 carbons. In certain embodiments of Formula 17, Z isacetoxymethyl.

In some embodiments of Formula 17, A is acetyl. In some embodiments ofFormula 17, R¹-R¹² are independently H, alkyl, aryl or halogen; R²⁰ andR²¹ are independently alkyl, aryl or heteroaryl; and Z is acetoxymethyl.

In some embodiments of Formula 17, A is acetyl; R¹-R¹² are independentlyH, alkyl, fluoro or chloro; R²⁰ and R²¹ are methyl; and Z isacetoxymethyl.

In some embodiments, the compound is described by Formula 18:

wherein R¹, R², R⁵, R⁶ and R¹⁰ are independently H, halogen, carboxy,alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso,cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid,aryl or heteroaryl; or alkyl, or alkoxy that is itself optionallysubstituted one or more times by halogen, amino, hydroxy, phosphonyl,sulfonyl, carbonyl, boronic acid, aryl or heteroaryl; R²⁰ and R²¹ areindependently alkyl, carboxyalkyl, aryl or heteroaryl; or alkyl, oralkoxy that is itself optionally substituted one or more times byhalogen, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid,aryl or heteroaryl; A is an acyl or an acyloxymethyl having 1-10carbons; and Z is an acyloxymethyl having 3-10 carbons.

In some embodiments of Formula 18, A is acetyl or acetoxymethyl. Incertain embodiments of Formula 18, A is acetyl and Z is an acyloxymethylhaving 3-10 carbons. In certain embodiments of Formula 18, Z isacetoxymethyl.

In some embodiments of Formula 18, A is acetyl. In some embodiments ofFormula 18, R¹, R², R⁵, R⁶ and R¹⁰ are independently H, alkyl, aryl orhalogen; R²⁰ and R²¹ are independently alkyl, aryl or heteroaryl; and Zis acetoxymethyl.

In some embodiments of Formula 18, A is acetyl; R¹, R², R⁵, R⁶ and R¹⁰are independently H, alkyl, fluoro or chloro; R²⁰ and R²¹ are methyl;and Z is acetoxymethyl.

In some embodiments, the compound is described by Formula 19:

wherein R¹, R², R⁵, R⁶ and R¹⁰ are independently H, halogen, carboxy,alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso,cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid,aryl or heteroaryl; or alkyl, or alkoxy that is itself optionallysubstituted one or more times by halogen, amino, hydroxy, phosphonyl,sulfonyl, carbonyl, boronic acid, aryl or heteroaryl; A is an acyl or anacyloxymethyl having 1-10 carbons; and Z is an acyloxymethyl having 3-10carbons.

In some embodiments of Formula 19, A is acetyl or acetoxymethyl. Incertain embodiments of Formula 19, A is acetyl and Z is an acyloxymethylhaving 3-10 carbons. In certain embodiments of Formula 19, Z isacetoxymethyl.

In some embodiments of Formula 19, A is acetyl. In some embodiments ofFormula 19, R¹, R², R⁵, R⁶ and R¹⁰ are independently H, alkyl, aryl orhalogen; and Z is acetoxymethyl.

In some embodiments of Formula 19, A is acetyl; R¹, R², R⁵, R⁶ and R¹⁰are independently H, alkyl, fluoro or chloro; and Z is acetoxymethyl. Insome embodiments of Formula 19, A is acetoxymethyl; R¹, R², R⁵, R⁶ andR¹⁰ are independently H, alkyl, fluoro or chloro; and Z isacetoxymethyl.

In some embodiments, the compound is described by Formula 20:

wherein A is acetyl or acetoxymethyl; R¹, R², R⁵ and R⁶ areindependently hydrogen, chloro or fluoro; and R¹⁰ is hydrogen, chloro,fluoro, nitro, methyl, ethyl, propyl, butyl, pentyl or hexyl.

In some embodiments of Formula 20, A is acetyl or acetoxymethyl. Incertain embodiments of Formula 20, A is acetyl. In certain embodimentsof Formula 20, A is acetoxymethyl.

In some embodiments of Formula 20, R² and R⁵ are chloro. In someembodiments of Formula 20, R¹ and R⁶ are chloro. In certain embodimentsof Formula 20, R¹, R², R⁵ and R⁶ are each chloro. In some embodiments ofFormula 20, R² and R⁵ are fluoro. In some embodiments of Formula 20, R¹and R⁶ are fluoro. In certain embodiments of Formula 20, R¹, R², R⁵ andR⁶ are each fluoro.

In some embodiments of Formula 20, R¹⁰ is hydrogen. In certainembodiments of Formula 20, R¹⁰ is methyl.

In certain embodiments of Formula 20, A is acetyl, R² and R⁵ are chloro,R¹ and R⁶ are hydrogen, and R¹⁰ is methyl.

In certain embodiments of Formula 20, A is acetyl, R² and R⁵ are fluoro,R¹ and R⁶ are hydrogen, and R¹⁰ is methyl.

In certain embodiments of Formula 20, A is acetyl, R² and R⁵ are chloro,R¹ and R⁶ are chloro, and R¹⁰ is hydrogen.

In certain embodiments of Formula 20, A is acetoxymethyl, R² and R⁵ arechloro, R¹ and R⁶ are hydrogen, and R¹⁰ is methyl.

In another aspect of the invention, the compounds of the invention maybe described by Formula 7:

In this embodiment, substituents R¹-R¹² are independently H, halogen,carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro,nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronicacid, aryl or heteroaryl; or alkyl, or alkoxy that is itself optionallysubstituted one or more times by halogen, amino, hydroxy, phosphonyl,sulfonyl, carbonyl, boronic acid, aryl or heteroaryl; R²⁰ and R²¹ areindependently alkyl, carboxyalkyl, aryl or heteroaryl; or alkyl, oralkoxy that is itself optionally substituted one or more times byhalogen, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid,aryl or heteroaryl; A is an alkyl having 1-10 carbons. In certainembodiments, A is methyl; Z is an acyloxymethyl having 3-10 carbons. Incertain embodiments, Z is acetoxymethyl.

In another aspect of the invention, A is alkyl; R¹-R¹² are independentlyH, alkyl, aryl or halogen; R²⁰ and R²¹ are independently alkyl, aryl orheteroaryl; Z is acetoxymethyl.

In another aspect of the invention, A is methyl; R¹-R¹² areindependently H, alkyl, fluoro or chloro; R²⁰ and R²¹ are methyl; Z isacetoxymethyl.

In another aspect of the invention, the compounds of the invention maybe described by Formula 8:

In this embodiment, substituents R¹-R¹² are independently H, halogen,carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro,nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronicacid, aryl or heteroaryl; or alkyl, or alkoxy that is itself optionallysubstituted one or more times by halogen, amino, hydroxy, phosphonyl,sulfonyl, carbonyl, boronic acid, aryl or heteroaryl; R²⁰ and R²¹ areindependently alkyl, carboxyalkyl, aryl or heteroaryl; or alkyl, oralkoxy that is itself optionally substituted one or more times byhalogen, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid,aryl or heteroaryl; R²⁴-R²⁹ are independently hydrogen, alkyl,carboxyalkyl, aryl or heteroaryl; or alkyl, or alkoxy that is itselfoptionally substituted one or more times by halogen, amino, hydroxy,phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or heteroaryl; A isan alkyl having 1-10 carbons. In certain embodiments, A is methyl; Z isan acyloxymethyl having 3-10 carbons.

In another aspect of the invention, A is alkyl; R¹-R¹² are independentlyH, alkyl, aryl or halogen; R²⁰ and R²¹ are independently alkyl, aryl orheteroaryl; R²⁴-R²⁹ are independently hydrogen, alkyl or aryl; Z is anacyloxymethyl having 3-10 carbons.

In another aspect of the invention, A is methyl; R¹-R¹² areindependently H, alkyl, fluoro or chloro; R²⁰ and R²¹ are methyl;R²⁴-R²⁹ are independently hydrogen or alkyl; Z is acetoxymethyl.

In another aspect of the invention, the compounds of the invention maybe described by Formula 9:

In this embodiment, substituents R¹-R¹² are independently H, halogen,carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro,nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronicacid, aryl or heteroaryl; or alkyl, or alkoxy that is itself optionallysubstituted one or more times by halogen, amino, hydroxy, phosphonyl,sulfonyl, carbonyl, boronic acid, aryl or heteroaryl; R²⁰-R²⁹ areindependently alkyl, carboxyalkyl, aryl or heteroaryl; or alkyl, oralkoxy that is itself optionally substituted one or more times byhalogen, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid,aryl or heteroaryl; A is an alkyl having 1-10 carbons. In certainembodiments, A is methyl; Z is an acyloxymethyl having 3-10 carbons.

In another aspect of the invention, A is alkyl; R¹-R¹² are independentlyH, alkyl, aryl or halogen; R²⁰ and R²¹ are independently alkyl, aryl orheteroaryl; R²⁴-R²⁹ are independently hydrogen, alkyl or aryl; Z is anacyloxymethyl having 3-10 carbons.

In another aspect of the invention, A is methyl; R¹-R¹² areindependently H, alkyl, fluoro or chloro; R²⁰ and R²¹ are methyl;R²⁴-R²⁹ are independently hydrogen or alkyl; Z is acetoxymethyl.

In another aspect of the invention, the compounds of the invention maybe described by Formula 10:

In this embodiment, substituents R¹-R⁸ are independently H, halogen,carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro,nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronicacid, aryl or heteroaryl; or alkyl, or alkoxy that is itself optionallysubstituted one or more times by halogen, amino, hydroxy, phosphonyl,sulfonyl, carbonyl, boronic acid, aryl or heteroaryl; R²⁰ and R²¹ areindependently alkyl, carboxyalkyl, aryl or heteroaryl; or alkyl, oralkoxy that is itself optionally substituted one or more times byhalogen, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid,aryl or heteroaryl; Z is an acyloxymethyl having 3-10 carbons.

In another aspect of the invention, A is alkyl; R¹-R⁸ are independentlyH, alkyl, aryl or halogen; R²⁰ and R²¹ are independently alkyl, aryl orheteroaryl; Z is an acyloxymethyl having 3-10 carbons.

In another aspect of the invention, A is methyl; R¹-R⁸ are independentlyH, alkyl, fluoro or chloro; R²⁰ and R²¹ are methyl; Z is acetoxymethyl.

In another aspect of the invention, the compounds of the invention maybe described by Formula 11:

In this embodiment, substituents R¹-R⁸ are independently H, halogen,carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro,nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronicacid, aryl or heteroaryl; or alkyl, or alkoxy that is itself optionallysubstituted one or more times by halogen, amino, hydroxy, phosphonyl,sulfonyl, carbonyl, boronic acid, aryl or heteroaryl; R²⁰ and R²¹ areindependently alkyl, carboxyalkyl, aryl or heteroaryl; or alkyl, oralkoxy that is itself optionally substituted one or more times byhalogen, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid,aryl or heteroaryl; A is an alkyl having 1-10 carbons. In certainembodiments, A is methyl; Z is an acyloxymethyl having 3-10 carbons.

In another aspect of the invention, A is alkyl; R¹-R⁸ are independentlyH, alkyl, aryl or halogen; R²⁰ and R²¹ are independently alkyl, aryl orheteroaryl; Z is an acyloxymethyl having 3-10 carbons.

In another aspect of the invention, A is methyl; R¹-R⁸ are independentlyH, alkyl, fluoro or chloro; R²⁰ and R²¹ are methyl; Z is acetoxymethyl.

In another aspect of the invention, the compounds of the invention maybe described by Formula 12:

In this embodiment, substituents R¹-R⁸ are independently H, halogen,carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro,nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronicacid, aryl or heteroaryl; or alkyl, or alkoxy that is itself optionallysubstituted one or more times by halogen, amino, hydroxy, phosphonyl,sulfonyl, carbonyl, boronic acid, aryl or heteroaryl; R²⁰ and R²¹ areindependently alkyl, carboxyalkyl, aryl or heteroaryl; or alkyl, oralkoxy that is itself optionally substituted one or more times byhalogen, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid,aryl or heteroaryl; Z is an acyloxymethyl having 3-10 carbons.

In another aspect of the invention, A is alkyl; R¹-R⁸ are independentlyH, alkyl, aryl or halogen; R²⁰ and R²¹ are independently alkyl, aryl orheteroaryl; Z is an acyloxymethyl having 3-10 carbons.

In another aspect of the invention, A is methyl; R¹-R⁸ are independentlyH, alkyl, fluoro or chloro; R²⁰ and R²¹ are methyl; Z is acetoxymethyl.

In another aspect of the invention, the compounds of the invention maybe described by Formula 13:

In this embodiment, substituents R¹-R¹² are independently H, halogen,carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro,nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronicacid, aryl or heteroaryl; or alkyl, or alkoxy that is itself optionallysubstituted one or more times by halogen, amino, hydroxy, phosphonyl,sulfonyl, carbonyl, boronic acid, aryl or heteroaryl; R²⁰ and R²¹ areindependently alkyl, carboxyalkyl, aryl or heteroaryl; or alkyl, oralkoxy that is itself optionally substituted one or more times byhalogen, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid,aryl or heteroaryl; R²⁴ and R²⁵ are independently hydrogen, alkyl,carboxymethyl or acetoxymethoxylenecarbonylmethyl; A is an alkyl having1-10 carbons. In certain embodiments, A is methyl; Z is an acyloxymethylhaving 3-10 carbons. In certain embodiments, Z is acetoxymethyl.

In another aspect of the invention, A is alkyl; R¹-R¹² are independentlyH, alkyl, aryl or halogen; R²⁰ and R²¹ are independently alkyl, aryl orheteroaryl; R²⁴ and R²⁵ are independently hydrogen, alkyl oracetoxymethoxylenecarbonylmethyl; A is methyl; Z is acetoxymethyl.

In another aspect of the invention, A is methyl; R¹-R¹² areindependently H, alkyl, fluoro or chloro; R²⁰ and R²¹ are methyl; R²⁴and R²⁵ are independently hydrogen, or acetoxymethoxylenecarbonylmethyl;R²⁴ and R²⁵ are a acetoxymethoxylenecarbonylmethyl; Z is acetoxymethyl.

In another aspect of the invention, the compounds of the invention maybe described by Formula 14:

In this embodiment, substituents R¹-R⁸ are independently H, halogen,carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro,nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronicacid, aryl or heteroaryl; or alkyl, or alkoxy that is itself optionallysubstituted one or more times by halogen, amino, hydroxy, phosphonyl,sulfonyl, carbonyl, boronic acid, aryl or heteroaryl; R²⁰ and R²¹ areindependently alkyl, carboxyalkyl, aryl or heteroaryl; or alkyl, oralkoxy that is itself optionally substituted one or more times byhalogen, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid,aryl or heteroaryl; A is an alkyl having 1-10 carbons. In certainembodiments, A is methyl.

In another aspect of the invention, A is alkyl; R¹-R⁸ are independentlyH, alkyl, aryl or halogen; R²⁰ and R²¹ are independently alkyl, aryl orheteroaryl.

In another aspect of the invention, A is methyl; R¹-R⁸ are independentlyH, alkyl, fluoro or chloro; R²⁰ and R²¹ are methyl.

In another aspect of the invention, the compounds of the invention maybe described by Formula 15:

In this embodiment, substituents R¹-R⁸ are independently H, halogen,carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro,nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronicacid, aryl or heteroaryl; or alkyl, or alkoxy that is itself optionallysubstituted one or more times by halogen, amino, hydroxy, phosphonyl,sulfonyl, carbonyl, boronic acid, aryl or heteroaryl; R²⁰ and R²¹ areindependently alkyl, carboxyalkyl, aryl or heteroaryl; or alkyl, oralkoxy that is itself optionally substituted one or more times byhalogen, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid,aryl or heteroaryl; A is an alkyl having 1-10 carbons. In certainembodiments, A is methyl.

In another aspect of the invention, A is alkyl; R¹-R⁸ are independentlyH, alkyl, aryl or halogen; R²⁰ and R²¹ are independently alkyl, aryl orheteroaryl.

In another aspect of the invention, A is methyl; R¹-R⁸ are independentlyH, alkyl, fluoro or chloro; R²⁰ and R²¹ are methyl.

In another aspect of the invention, the compounds of the invention maybe described by Formula 15:

In this embodiment, substituents R¹-R⁸ are independently H, halogen,carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro,nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronicacid, aryl or heteroaryl; or alkyl, or alkoxy that is itself optionallysubstituted one or more times by halogen, amino, hydroxy, phosphonyl,sulfonyl, carbonyl, boronic acid, aryl or heteroaryl; R²⁰ and R²¹ areindependently alkyl, carboxyalkyl, aryl or heteroaryl; or alkyl, oralkoxy that is itself optionally substituted one or more times byhalogen, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid,aryl or heteroaryl; R²⁴ is hydrogen, alkyl, carboxymethyl oracetoxymethoxylenecarbonylmethyl; A is an alkyl having 1-10 carbons.

In another aspect of the invention, A is methyl; R¹-R⁸ are independentlyH, alkyl, aryl or halogen; R²⁰ and R²¹ are independently alkyl, aryl orheteroaryl; R²⁴ is hydrogen, alkyl, carboxymethyl oracetoxymethoxylenecarbonylmethyl.

In another aspect of the invention, A is methyl; R¹-R⁸ are independentlyH, alkyl, fluoro or chloro; R²⁰ and R²¹ are methyl; R²⁴ isacetoxymethoxylenecarbonylmethyl.

In another aspect of the invention, the compounds of the invention maybe described by Formula 16:

In this embodiment, substituents R¹-R⁸ are independently H, halogen,carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro,nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronicacid, aryl or heteroaryl; or alkyl, or alkoxy that is itself optionallysubstituted one or more times by halogen, amino, hydroxy, phosphonyl,sulfonyl, carbonyl, boronic acid, aryl or heteroaryl; R²⁰ and R²¹ areindependently alkyl, carboxyalkyl, aryl or heteroaryl; or alkyl, oralkoxy that is itself optionally substituted one or more times byhalogen, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid,aryl or heteroaryl; A is an alkyl having 1-10 carbons; R²⁴ and R²⁵ areindependently hydrogen, alkyl, carboxymethyl oracetoxymethoxylenecarbonylmethyl.

In another aspect of the invention, A is methyl; R¹-R⁸ are independentlyH, alkyl, aryl or halogen; R²⁰ and R²¹ are independently alkyl, aryl orheteroaryl; R²⁴ and R²⁵ are independently hydrogen, alkyl, carboxymethylor acetoxymethoxylenecarbonylmethyl.

In another aspect of the invention, A is methyl; R¹-R⁸ are independentlyH, alkyl, fluoro or chloro; R²⁰ and R²¹ are methyl; R²⁴ and R²⁵ areacetoxymethoxylenecarbonylmethyl.

The fluorophore moiety can be any compound described by any of Formulas1-16 that exhibits an absorption maximum beyond 500 nm uponesterase-induced hydrolysis, that is fused to a chelator, as describedin greater detail below.

In one aspect of the invention, the fluorophore moiety has an absorptionmaximum beyond 500 nm. The fluorophore moiety is typically selected toconfer its fluorescence properties on the indicator compound it isincorporated into. That is, the resulting indicator compound exhibits adetectable optical response when excited by energy having a wavelengthat which that fluorophore absorbs as used herein, a detectable opticalresponse means a change in, or occurrence of, an optical property thatis detectable either by observation or instrumentally, such a change inabsorption (excitation) wavelength, fluorescence emission wavelength,fluorescence emission intensity, fluorescence polarization, orfluorescence lifetime, among others.

In addition, the compounds of the invention preferably exhibit adetectable change in the optical response upon binding a target metalion. Where the detectable response is a fluorescence response, thedetectable change is typically a change in fluorescence, such as achange in the intensity, excitation or emission wavelength distributionof fluorescence, fluorescence lifetime, fluorescence polarization, or acombination thereof. In certain embodiments, the change in opticalresponse upon binding the target metal ion is a change in fluorescenceintensity that is greater than approximately 50-fold, more preferablygreater than 100-fold.

Synthesis

The compounds of the invention may be prepared using any suitablesynthetic schemes. The methodology used to prepare the compounds of theinvention may involve a few components. The first component may involvethe formation of the chelator, while the second may involve themodification of the chelator by forming a reactive functional group,covalently attaching a conjugate, or covalently attaching a fluorophoremoiety to form the desired indicator compound. Although these syntheticcomponents are typically performed in the order given, they may becarried out in any other suitable sequence. For example, a portion ofthe chelator may be derivatized with a fluorescent dye prior toformation of the complete chelator ring. The representative syntheticmethods are summarized below. The other appropriate methods may also beadapted to synthesize the desired compounds of the invention.

As the metal binding ability of the resulting chelators may besignificantly influenced by the nature of the amine substituents,careful selection of the alkylating agent may be necessary to prepare areporter for a particular target ion. BAPTA chelators are typicallyselective for calcium ion. Where the chelator nitrogens are alkylated bymethyl bromoacetate, the resulting bis-aza-crown ether is typicallyselective for sodium ions. If the alkylating agent is 2-picolylchloride, the resulting crown ether is typically selective for zincions. EDTA derivatives are typically used for transitional metal ions.Selection of an alkylating agent that incorporates a precursor to areactive functional group is useful for producing chemically reactivecompounds of the invention, as well as acting as a useful intermediatefor preparing conjugates, as described above.

The syntheses of chelating groups selective for different metal ions hasbeen well described in the literature (U.S. Pat. No. 4,603,209; U.S.Pat. No. 5,049,673; U.S. Pat. No. 4,849,362; U.S. Pat. No. 5,453,517;U.S. Pat. No. 5,501,980; U.S. Pat. No. 5,459,276; U.S. Pat. No.5,501,980; U.S. Pat. No. 5,459,276; U.S. Pat. No. 5,516,911; U.S.Application No. 2002/0164616; each of which is incorporated byreference). These methods can be readily adapted to prepare chelatorintermediates useful for the synthesis of the compounds of theinvention.

Synthesis of carbofluorescein dyes typically involves the condensationof properly substituted bisphenol with a carbonyl-containing moiety suchas a phthalic acid derivative or benzaldehyde derivatives. In thesynthesis of the carbofluorescein indicators of the invention, thedesired bisphenol is condensed with a chelator intermediate thatcontains a carboxylic acid, anhydride or acyl halide bound directly tothe chelating moiety. This synthetic method is illustrated in Scheme 1.

Under acidic conditions, the synthesis of carbofluorescein dyes mightalso be achieved from the condensation of properly substituted bisphenolwith a 2-carboxybenzaldehyde-containing moiety followed by airoxidation. In the synthesis of the carbofluorescein indicators of theinvention, the desired bisphenol is condensed with a chelatorintermediate that contains a 2-carboxybenzaldehyde bound directly to thechelating moiety. This synthetic method is illustrated in Scheme 2.

The condensation of a properly substituted benzophenone with a desired3-hydroxymethylphenol derivative can also provide a good access tocarbofluorescein dyes. In this synthesis 3-hydroxyphenylstyryl can be agood alternative to the synthon of 3-hydroxymethylphenol derivative.This synthetic method is illustrated in Schemes 3 and 4.

Alternatively the fluorescent indicators of the invention can beprepared via the condensation of properly protected xanthones with achelator anion, typically prepared from the corresponding chelatorbromide or iodide. This organometallic chemistry is also well describedin the literature (C. Chen, R. Yeh and D. S. Lawrence, J. Am. Chem. Soc.2002, 124, 3840; U.S. Pat. No. 5,049,673; Y. Urano, M. Kamiya, K. Kanda,T. Ueno, K. Hirose and T. Nagano, J. Am. Chem. Soc. 2005, 127, 4888) andcan be readily adapted to synthesize the compounds of the invention. Theprotection and deprotection conditions are well described in theliterature (C. J. Kocienski, Protecting Groups, 3^(rd) Ed, Georg ThiemeVerlag, 2005, pp 187-364 and pp 393-450; T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 3^(rd) Ed, John Wiley & Sons,1999, pp 246-292 and pp 369-453). This synthetic method is illustratedin Scheme 5.

Post-condensation modifications of both the chelator and the fluorophoremoiety are typically analogous to known methods of indicatormodification. For example, the reduction of nitro substituents to aminogroups, the conversion of carboxy substituents to cyano groups, and thepreparation of esters of carboxylic acids, including acetoxymethylesters. Additionally, a given salt or counterion of the indicators ofthe invention may be readily converted to other salts by treatment withion-exchange resins, selective precipitation, and basification, as iswell-known in the art.

Post-condensation modifications of phenol dyes are well known. Forinstance, the xanthenone portion of the dye can be halogenated bytreatment with an appropriate halogenating agent, such as NCS,hypochlorite, sulfuryl chloride, bromine, NBS and iodine. Xanthenescontaining unsaturated fused rings can be hydrogenated to the saturatedderivatives.

The reduced and oxidized versions of the carbofluorescein indicators arefreely interconvertible by well-known oxidation or reduction reagents,including borohydrides, aluminum hydrides, hydrogen/catalyst, anddithionites. Care must be exercised to select an oxidation or reducingagent that is compatible with the chelator used. A variety of oxidizingagents mediate the oxidation of dihydroxanthenes, including molecularoxygen in the presence or absence of a catalyst, nitric oxide,peroxynitrite, dichromate, triphenylcarbenium and chloranil. The dihydrocarbofluoresceins may also be oxidized electrochemically, or by enzymeaction, including the use of horseradish peroxidase in combination withperoxides or by nitric oxide.

Applications of the Fluorescent Indicators of the Invention

The indicators disclosed herein possess particular utility for thedetection and/or quantification of metal ions in a sample of interest.Such indicators may be useful for measuring ions in extracellularspaces; in vesicles; in vascular tissue of plants and animals;biological fluids such as blood and urine; in fermentation media; inenvironmental samples such as water, soil, waste water and seawater; andin chemical reactors. Optical indicators for ions are important forqualitative and quantitative determination of ions, particularly inliving cells. Fluorescent indicators for metal cations also permit thecontinuous or intermittent optical determination of these ions in livingcells, and in solutions containing the ions.

In effecting such determination, the substance to be determined, oranalyte, which contains the ion of interest is contacted with afluorescent indicator as disclosed above. Complexation of the metal ionin the chelator of the indicator results in a detectable change in thefluorescence properties of the indicator. Detection and optionallyquantification of the detectable change permits the ion of interest tobe detected and optionally quantified.

Upon binding the target ion in the chelating moiety of the indicator,the optical properties of the attached fluorophore are generallyaffected in a detectable way, and this change may be correlated with thepresence of the ion according to a defined standard. Compounds havingrelatively long wavelength excitation and emission bands can be usedwith a variety of optical devices and require no specialized (quartz)optics, such as are required by indicators that are excited or that emitat shorter wavelengths. These indicators are suitable for use influorescence microscopy, flow cytometry, fluorescence microplatereaders, or any other application that currently utilize fluorescentmetal ion indicators.

This determination method may be based on the so-called “PET effect”, orthe transfer, induced by photons, of electrons (photoinduced electrontransfer=PET) from the ionophoric moiety or ionophore, respectively, tothe fluorophore moiety or fluorophore, respectively, which leads to adecrease in the (relative) fluorescence intensity and the fluorescencedecay time of the fluorophore. Absorption and emission wavelengths,however, are not significantly affected in the process (J. R. Lakowiczin “Topics in Fluorescence Spectroscopy”, Volume 4: Probe Design andChemical Sensing; Plenum Press, New York & London (1994)).

By the binding of ions to the ionophore, the PET effect may be partly orcompletely inhibited, so that there is an increase in the fluorescenceof the fluorophore moiety. Hence, the concentration or the activity ofthe ion to be determined can be deduced by measuring the change influorescence properties, i.e. fluorescence intensity and/or fluorescencedecay time.

A variety of fluorescent indicators that are useful for the detection ofbiologically relevant soluble free metal ions (such as Ca²⁺, Mg²⁺ andZn²⁺) have been described that utilize oxygen-containing anionic orpolyanionic chelators to bind to metal ions. In general, a usefulproperty for metal ion indicators is selectivity, or the ability todetect and/or quantify a selected metal ion in the presence of othermetal ions. Discrimination of Ca²⁺, Na⁺ and K⁺ ions in the presence ofother metal ions is particularly advantageous in certain biological orenvironmental samples. For most biological applications, it is usefulthat the indicators be effective in aqueous solutions. It is alsobeneficial if the indicator absorbs and emits light in the visiblespectrum where biological materials typically have low intrinsicabsorbance or fluorescence.

Optical methods using fluorescence detection of metal ions permitmeasurement of the entire course of ion flux in a single cell as well asin groups of cells. The advantages of monitoring transport byfluorescence techniques include the high level of sensitivity of thesemethods, temporal resolution, modest demand for biological material,lack of radioactivity, and the ability to continuously monitor iontransport to obtain kinetic information (Eidelman, O. Cabantchik, Z. I.Biochim. Biophys. Acta, 1989, 988, 319-334). The general principle ofmonitoring transport by fluorescence is based on havingcompartment-dependent variations in fluorescence properties associatedwith translocation of compounds.

Optical methods were developed initially for measuring Ca²⁺ ion flux(U.S. Pat. No. 5,049,673; Scarpa, A. Methods of Enzymology, 1979, 56,301 Academic Press, Orlando, Fla.; Tsien, R. Y. Biochemistry, 1980, 19,2396; Grynkiewicz, G., Poenic, M., Tsien, R. Y. J. Biol Chem., 260,3440) and have been modified for high-throughput assays (U.S. Pat. No.6,057,114). The flux of Ca²⁺ ion is typically performed usingcalcium-sensitive fluorescent dyes such as Fluo-3, Fluo-4, Fluo-8,Cal-520, Calcium Green, and others.

In particular, fluorescent indicators utilizing a BAPTA chelator havebeen previously described. A determination method utilizingaza-cryptands as the chelator moiety and using xanthenes and coumarinsas fluorophores has also been described (U.S. Pat. No. 5,439,828 and USPatent Application 20020164616). These aza-cryptand may, depending ontheir structure, exhibit selectivity for lithium, sodium or potassiumions. Some fluorescent indicators selective for Li⁺, Na⁺ and K⁺ inaqueous or organic solution have also been described, based on thechemical modification of crown ethers (U.S. Pat. No. 5,134,232; U.S.Pat. No. 5,405,975).

A variety of carbofluorescein metal ion indicators can be prepared asdiscussed above. A person skilled in the art can readily preparecarbofluorescein indicators using the methods discussed above. Selectedembodiments of the invention are given in Table 4 for illustrationpurpose:

TABLE 1 Example compounds of the invention: Indicator Structure Use  8

Detecting Ca²⁺  9

Detecting Ca²⁺ 16

Detecting Ca²⁺ 85

Detecting Ca²⁺ 93

Detecting Zn²⁺ and Mg²⁺ 125

Detecting Ca²⁺ 128

Detecting Ca²⁺ 139

Detecting transitional metal ions 145

Detecting Na⁺ 150

Detecting Ni²⁺ 200

Detecting Ca²⁺ 201

Detecting Ca²⁺ 202

Detecting Ca²⁺ 203

Detecting Ca²⁺ 204

Detecting Zn²⁺ and Mg²⁺ 205

Detecting Ca²⁺ 206

Detecting Ca²⁺ 207

Detecting transitional metal ions 208

Detecting Na⁺ 209

Detecting transitional metal ions 210

Detecting Ca²⁺ 211

Detecting Zn²⁺ 212

Detecting Ca²⁺ 213

Detecting Ca²⁺ 214

Detecting Mg²⁺ 215

Detecting Ca²⁺ 216

Detecting Ca²⁺ 217

Detecting Ca²⁺ 218

Detecting Ca²⁺ 219

Detecting Ca²⁺ 220

Detecting Ca²⁺ 221

Detecting Ca²⁺ 222

Detecting Ca²⁺ 223

Detecting Ca²⁺ 224

Detecting Ca²⁺ 225

Detecting Ca²⁺ 226

Detecting Ca²⁺ 227

Detecting Ca²⁺ 228

Detecting Ca²⁺ 229

Detecting Ca²⁺ 230

Detecting Ca²⁺ 231

Detecting Ca²⁺ 232

Detecting Ca²⁺ 233

Detecting Ca²⁺ 234

Detecting Ca²⁺ 235

Detecting Ca²⁺ 236

Detecting Ca²⁺ 237

Detecting Ca²⁺ 238

Detecting Ca²⁺ 239

Detecting Ca²⁺ 240

Detecting Ca²⁺ 241

Detecting Ca²⁺

The desired indicator compound is generally prepared for use as adetection reagent by dissolving the indicator in solution at aconcentration that is optimal for detection of the indicator at theexpected concentration of the target ion. Modifications that aredesigned to enhance permeability of the indicator through the membranesof live cells, such as functionalization of carboxylic acid moietiesusing acetoxymethyl esters and acetates, may require the indicator to bepredissolved in an organic solvent such as dimethylsulfoxide (DMSO)before addition to a cell suspension, where the indicators may thenreadily enter the cells. Intracellular enzymes then cleave the esters,generating more polar acids and phenols which are then well-retainedinside the cells. For applications where permeability of cell-membranesis required, the indicators of the invention are typically substitutedby only one fluorophore.

The specific indicator used in a particular assay or experiment may beselected based on the desired affinity for the target ion as determinedby the expected concentration range in the sample, the desired spectralproperties, and the desired selectivity. Initially, the suitability of amaterial as an indicator of ion concentration is commonly tested bymixing a constant amount of the indicating reagent with a measuredamount of the target ion under the expected experimental conditions.

Where the binding of an ion in the metal ion-binding moiety of theindicator results in a detectable change in spectral properties of theindicator compound, that indicator may be used for the detection and/orquantification of that ion (the target ion). Although the change inspectral properties may include for example a change in absorptionintensity or wavelength, preferably the change in spectral properties isa detectable fluorescence response. Preferred indicators display a highselectivity, that is, they show a sufficient rejection of non-targetions. The interference of a non-target ion is tested by a comparabletitration of the indicator with that ion. In one aspect of theinvention, the target ions for the indicators of the present inventionare selected from Ca²⁺, Na⁺ and K⁺.

A detectable fluorescence response, as used herein, is a change in afluorescence property of the indicator that is capable of beingperceived, either by direct visual observation or instrumentally, thepresence or magnitude of which is a function of the presence and/orconcentration of a target metal ion in the test sample. This change in afluorescence property is typically a change in fluorescence quantumyield, fluorescence polarization, fluorescence lifetime, a shift inexcitation or emission wavelength, among others, or a combination of oneor more of such changes in fluorescence properties. The detectablechange in a given spectral property is generally an increase or adecrease. However, spectral changes that result in an enhancement offluorescence intensity and/or a shift in the wavelength of fluorescenceemission or excitation may also be useful. The change in fluorescence onion binding may be due to conformational or electronic changes in theindicator that may occur in either the excited or ground state of thefluorophore, due to changes in electron density at the ion binding site,due to quenching of fluorescence by the bound target metal ion, or dueto any combination of these or other effects.

A typical indicator for a specific target ion is an indicator thatexhibits at least a 50-fold change in net fluorescence emissionintensity (either an increase or decrease), or at least a 1 nanoseconddifference in fluorescence lifetime (either shorter or longer). In oneaspect of the invention, the indicator exhibits a 50-fold or greaterchange in net fluorescence emission intensity, and/or a 100% change influorescence lifetime in the presence of the target ion.

The spectral response of a selected indicator to a specific metal ion isa function of the characteristics of the indicator in the presence andabsence of the target ion. For example, binding to a metal ion may alterthe relative electron densities of the fluorophore and the metal bindingsite. Additionally, or in the alternative, some metal ions may quenchfluorescence emission when in close proximity to a fluorophore (heavyatom quenching). In one embodiment of the invention, the indicator isessentially nonfluorescent or exhibits low fluorescence in targetion-free solution and exhibits an increase in fluorescence intensity orfluorescence lifetime (or both) upon target metal ion binding.

As the optical response of the indicating reagent is typicallydetermined by changes in fluorescence, the threshold of detection of thetarget ion will be dependent upon the sensitivity of the equipment usedfor its detection.

If the optical response of the indicator will be determined usingfluorescence measurements, the sample of interest is typically stainedwith indicator concentrations of 10⁻⁹ M to 10⁻³ M. The most useful rangeof analyte concentration includes about one log unit above and below thedissociation constant of the ion-indicator complex. This dissociationconstant may be determined by titration of the indicator with knownconcentrations of the target ion, usually over the range of virtuallyzero concentration to approximately 100 mM of the target ion, dependingon which ion is to be measured and which indicator is being used. Thedissociation constant may be affected by the presence of other ions,particularly ions that have similar ionic radii and charge. It may alsobe affected by other conditions such as ionic strength, pH, temperature,viscosity, presence of organic solvents and incorporation of the sensorin a membrane or polymeric matrix, or conjugation or binding of thesensor to a protein or other biological molecule. Any or all of theseeffects are readily determined, and can be taken into account whencalibrating a selected indicator.

The indicator is typically combined with a sample in a way that willfacilitate detection of the target ion concentration in the sample. Thesample is generally a fluid or liquid suspension that is known orsuspected to contain the target ion. Representative samples includeintracellular fluids from cells such as in blood cells, cultured cells,muscle tissue, neurons and the like; extracellular fluids in areasimmediately outside of cells; fluids in vesicles; fluids in vasculartissue of plants and animals; biological fluids such as blood, saliva,and urine; biological fermentation media; environmental samples such aswater, soil, waste water and sea water; industrial samples such aspharmaceuticals, foodstuffs and beverages; and samples from chemicalreactors. Detection and quantitation of the target ion in a sample canhelp characterize the identity of an unknown sample, or facilitatequality control of a sample of known origin.

In one embodiment of the invention, the sample includes cells, and theindicator is combined with the sample in such a way that the indicatoris added within the sample cells. By selection of the appropriatechelating moiety, fluorophore, and the substituents thereon, indicatorsmay be prepared that will selectively localize in a desired organelle,and provide measurements of the target ion in those organelles.Conjugates of the indicators of the invention with organelle-targetingpeptides may be used to localize the indicator to the selectedorganelle, facilitating measurement of target ion presence orconcentration within the organelle (as described in U.S. Pat. No.5,773,227, hereby incorporated by reference). Alternatively, selectionof a lipophilic fluorophore, or a fluorophore having predominantlylipophilic substituents may result in localization of the indicator inlipophilic environments in the cell, such as cell membranes. Selectionof cationic indicators will typically result in localization of theindicator in mitochondria.

In one embodiment of the invention, the indicator compound of theinvention optionally further includes a metal ion. In anotherembodiment, the compounds of the invention, in any of the embodimentsdescribed above, are associated, either covalently or noncovalently,with a surface such as a microfluidic chip, a silicon chip, a microscopeslide, a microplate well, or another solid or semisolid matrix, and iscombined with the sample of interest as it flows over the surface. Inthis embodiment, the detectable optical response may therefore bedetected on the matrix surface itself, typically by use of instrumentaldetection. This embodiment of the invention may be particularly suitedto high-throughput screening using automated methods.

The fluorescence response of the indicator to the target ion may bedetected by various means that include without limitation measuringfluorescence changes with fluorometers, fluorescence microscopes, laserscanners, flow cytometers, and microfluidic devices, as well as bycameras and other imaging equipment. These measurements may be maderemotely by incorporation of the fluorescent ion sensor as part of afiber optic probe. The indicator may be covalently attached to the fiberoptic probe material, typically glass or functionalized glass (e.g.,aminopropyl glass) or the indicator may be attached to the fiber opticprobe via an intermediate polymer, such as polyacrylamide. The indicatorsolution is alternatively incorporated non-covalently within a fiberoptic probe, as long as there is a means whereby the target ion may comeinto contact with the indicator solution. More preferably, the BAPTAindicators of the invention are used with a fluorescence microplatereader that is equipped with an automated liquid handling system such asFLIPR, FlexStation and FDSS.

In another aspect of the invention, the fluorescent ion indicators ofthe invention may be used in combination with one or morenon-fluorescent dyes that are not substantially cell-permeable in orderto reduce the background fluorescence analogous to the methods describedin U.S. Pat. No. 6,420,183, hereby incorporated by reference.

Non-fluorescent dyes and dye mixtures that have large water solubilitiesand minimal effects on the physiology of the cells are preferred forthis application. More preferably are water-soluble azo dyes (such astrypan blue), which have been used in cell-based assays for many years(H. W. Davis, R. W. Sauter. Histochemistry, 1977, 54, 177; W. E.Hathaway, L. A. Newby, J. H. Githens, Blood, 1964, 23, 517; C. W. Adams,O. B. Bayliss, R. S. Morgan, Atherosclerosis, 1977, 27, 353).

The screening methods described herein can be performed with cellsgrowing in or deposited on solid surfaces. A common technique is to usea microwell plate where the fluorescence measurements are performingusing a commercially available fluorescent plate reader. These methodslend themselves to use in high throughput screening using both automatedand semi-automated systems.

Using the indicators of the present invention, the measurement offluorescence intensity can provide a sensitive method for monitoringchanges in intracellular ion concentrations. Thus, fluorescencemeasurements at appropriate excitation and emission wavelengths providea fluorescence readout which is sensitive to the changes in the ionconcentrations.

In one embodiment, the invention includes a) adding a compound asdescribed above to a sample containing a cell; b) incubating the samplefor a time sufficient for the compound to be loaded into the cell and anindicator compound to be generated intracellularly; c) illuminating thesample at a wavelength that generates a fluorescence response from theindicator compound; d) detecting a fluorescence response from theindicator compound; and e) correlating the fluorescence response withthe presence of intracellular calcium.

In one aspect of the invention, the disclosed method is useful forscreening potential therapeutic drugs, for example drugs which mayaffect ion concentrations in biological cells. These methods may includemeasuring ion concentrations as described above in the presence andabsence (as a control measurement) of the test sample. Controlmeasurements are usually performed with a sample containing allcomponents of the test sample except for the putative drug beingscreened. Detection of a change in ion concentration in the presence ofthe test agent relative to the control indicates that the test agent isactive. Ion concentrations can also be determined in the presence orabsence of a pharmacologic agent of known activity (i.e., a standardagent) or putative activity (i.e., a test agent). A difference in ionconcentration as detected by the methods disclosed herein allows one tocompare the activity of the test agent to that of a standard agent ofknown activity. It will be recognized that many combinations andpermutations of drug screening protocols are known to one of skill inthe art and they may be readily adapted to use with the method of ionconcentration measurement disclosed herein to identify compounds whichaffect ion concentrations.

Fluo-3 AM and Fluo-4 AM are predominantly used for monitoringintracellular calcium signal. However, Fluo-3 AM, Fluo-4 AM and theircell-hydrolyzed products (Fluo-3 and Fluo-4) can be simultaneouslyexcited by the same excitation source (see FIGS. 7 and 8), causing highassay background. The present application is directed to a family offluorescent dyes that are useful for preparing fluorescent metal ionindicators. The indicators include a carbofluorescein lactonefluorophore that is incorporated with an ionophore, and are useful forthe detection, discrimination and quantification of metal cations. Thefluorescent indicators of this invention demonstrate unexpected largerspectral shift upon cell-induced hydrolysis, as shown in Table 2, FIGS.7 and 8.

TABLE 2 Spectral shift comparison Cell-Induced Residual absorptionExcitation of dye AM Wavelength at the maximum Compound Shift (nm)excitation of dye acid Fluo-3 AM 36 nm 7.6% Compound 16 278 nm    0%Fluo-4 AM 38 nm 7.8% Compound 85 277 nm    0%

The fluorescent indicators of this invention demonstrate unexpectedemission shift to the longer wavelength as shown in Table 3. Thered-shift excitation and emission wavelengths provide a great advantagefor the use of current invention in the high throughput screening of newdrug candidates since there are many fluorescent compounds of shortwavelength in the screening compound libraries. Fluo-3 AM and Fluo-4 AMare currently used in the high throughput screening of new drugcandidates, but their short wavelength overlap with the fluorescence ofsome fluorescent compounds in the screening compound libraries, causingsevere interference.

TABLE 3 Spectral property comparison Excitation Emission CompoundWavelength (nm) Wavelength (nm) Fluo-3 504 nm 526 nm Compound 11 558 nm584 nm Fluo-4 493 nm 515 nm Compound 79 554 nm 582 nm

The fluorescent indicators of this invention demonstrate betterstability in the presence of cells and better cellular retentioncompared to the existing fluorescein ion indicators as shown in FIG. 2.The fluorescent indicators of this invention have doubleesterase-cleavable blocking groups that slow down the spontaneoushydrolysis in cell medium, thus reducing the assay background caused bythe spontaneously hydrolyzed indicators. In addition, the fluorescentindicators of this invention provide an extra negative charge upon cellhydrolysis, reducing the hydrolyzed indicators from leaking out ofcells, another factor that contributes the high assay background asshown in FIG. 9.

In one aspect of the invention, the disclosed calcium indicators havethe minimal assay background in their masked form since theirnon-hydrolyzed AM esters cannot be excited in the range of visiblewavelengths. As seen from FIG. 7, Compound 16 has essentially noabsorption at 555 nm, the optimal excitation wavelength used fordetecting intracellular calcium signal with Compound 16 while Fluo-3 AMhas substantial absorption at 488 nm, the optimal excitation wavelengthused for detecting intracellular calcium signal with Fluo-4 AM. Compound16 and Fluo-3 AM are the masked form that does not bind calcium, shallnot generate fluorescence signal unless binding calcium ion insidecells. The fluorescence outside cells generates the detrimental assaybackground. It is evident that the calcium indicators of the invention(e.g., Compound 16) have unexpected spectral properties that enable moresensitive detection of calcium in cells compared to the existingfluorescein-based calcium indicators (such as Fluo-3 AM).

As seen from FIG. 8, Compound 85 has essentially no absorption at 555 nm(the wavelength used to excite the deblocked Compound 85 inside cells)while Fluo-4 AM has substantial absorption at 488 nm (the wavelengthused to excite the deblocked Fluo-4 AM inside cells). Compound 85 andFluo-4 AM are the masked form that does not bind calcium, theirfluorescence caused by 555 nm (for Compound 85) and 488 nm (for Fluo-4)excitation generates the detrimental assay background. It is evidentthat the calcium indicators of the invention (e.g., Compound 85) haveunexpected spectral properties that enable more sensitive detection ofcalcium in cells compared to the existing fluorescein-based calciumindicators (such as Fluo-4 AM).

FIG. 5 indicates that Compound 79 (the hydrolyzed product of Compound85) binds calcium, and is well excited at 555 nm to give thefluorescence that is related to calcium concentration.

In yet another aspect of the invention, the fluorescent ion indicatorsare used in a method to measure calcium flux. Cells (e.g., CHO cells)stably transfected with muscarinic receptor 1 are plated—e.g., at 60,000cells per 100 μl per well in F12 with 5% FBS and 1% glutamine in a96-well black wall/clear bottom Costar plate—and incubated (e.g., in 5%CO₂ at 37° C. overnight). The growth medium is removed and the cells areincubated with a fluorescent ion indicator (e.g., with 100 μl/well of1-8 μM Fluo-4 AM or Compound 85 in Hanks and HEPES buffer for 1 hour atroom temperature) with or without probenecid. Carbachol is added (e.g.,50 μl/well by NOVOstar, FlexStation or FLIPR) to achieve a finalconcentration. Fluorescent ion indicators of the invention (e.g.,Compound 85) load into cells much better than Fluo-4 AM, at certain ATPconcentrations (e.g., 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM,0.7 μM, 0.8 μM, 0.9 μM, 1.0, 10 or 100 μM) loading more than 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% faster. When probenecid is notused, the fluorescence intensity of a fluorescent ion indicator of theinvention (e.g., Compound 85), is much greater than that of Fluo-4 AM.At certain ATP concentrations (e.g., 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0, 10 or 100 μM) the intensity ismore than 50%, 100%, 150%, 200%, 250%, 300%, 350% or 400% greater. Thecalcium indicators of the invention are unexpectedly well retainedinside cells compared to the existing fluorescein-based calciumindicators (such as Fluo-3 and Fluo-4) that quickly leaks out of cell,another factor resulting higher assay background besides their high 488nm absorption (see above). When probenecid is not used, Fluo-4 AM is notcapable of detecting calcium in some types of cells and tissues forwhich Compound 85 is used.

In yet another aspect of the invention, the disclosed method mayfacilitate the screening of test samples in order to identify one ormore compounds that are capable of modulating the activity of an ionchannel, pump or exchanger in a membrane, and the method furtherincludes stimulating the cell, monitoring changes in the intensity ofthe fluorescence response from the indicator compound, and correlatingthe changes in fluorescence intensity with changes in intracellularcalcium levels.

An additional method may be used to evaluate the efficacy of a stimulusthat generates a target ion response, including (a) loading a first setand a second set of cells with the ion indicators of the invention whichmonitor ion concentrations; (b) optionally, exposing both the first andsecond set of cells to a stimulus which modulates the ion channel, pumpor exchanger; (c) exposing the first set of cells to the test sample;(d) measuring the ion concentrations in the first and second sets ofcells; and (e) relating the difference in ion concentrations between thefirst and second sets of cells to the ability of a compound in the testsample to modulate the activity of an ion channel, pump or exchanger incells. In one aspect of the recited method, the method may include theaddition of probenecid or a probenecid derivative to the sample.

One or more of the methods disclosed herein may be enhanced by theaddition of a cell-impermeant and non-fluorescent dye to the sample,such that the dye remains in the extracellular solution, and acts as anacceptor dye for energy transfer from the indicator compound, therebydecreasing background signal from the sample solution. In one aspect ofthe method, the cell-impermeant and non-fluorescent dye is awater-soluble azo dye.

Ion channels of particular interest may include, but are not limited to,sodium, calcium, potassium, nonspecific cation, and chloride ionchannels, each of which may be constitutively open, voltage-gated,ligand-gated, or controlled by intracellular signaling pathways.

Biological cells of potential interest for screening application mayinclude, but are not limited to, primary cultures of mammalian cells,cells dissociated from mammalian tissue, either immediately or afterprimary culture. Cell types may include, but are not limited to whiteblood cells (e.g. leukocytes), hepatocytes, pancreatic beta-cells,neurons, smooth muscle cells, intestinal epithelial cells, cardiacmyocytes, glial cells, and the like. The disclosed method may alsoinclude the use of recombinant cells into which ion transporters, ionchannels, pumps and exchangers have been inserted and expressed bygenetic engineering. Many cDNA sequences for such transporters have beencloned (see U.S. Pat. No. 5,380,836 for a cloned sodium channel, herebyincorporated by reference) and methods for their expression in celllines of interest are within the knowledge of one of skill in the art(see, U.S. Pat. No. 5,436,128, hereby incorporated by reference).Representative cultured cell lines derived from humans and other mammalsinclude LM cells, HEK-293 (human embryonic kidney cells), 3T3fibroblasts, COS cells, CHO cells, RAT1 and HepG2 cells, Hela cells,U₂OS cells and Jurkat cells etc.

Assay Kits

Due to the advantageous properties and the simplicity of use of thedisclosed ion indicator compounds, they possess particular utility inthe formulation of a kit for the complexation, detection, orquantification of selected target ions. An exemplary kit may include oneor more compounds or compositions of the invention in any of theembodiments described above, either present as a pure compound, in asuitable composition, or dissolved in an appropriate stock solution. Thekit may further include instructions for the use of the indicatorcompound to complex or detect a desired target ion. The kit may furtherinclude one or more additional components, such as an additionaldetection reagent.

The indicator of the invention may be present in the kit associated witha surface, such as a chip, microplate well, or other solid or semi-solidmatrix.

The additional kit components may be selected from, without limitation,calibration standards of a target ion, ionophores, fluorescencestandards, aqueous buffers, surfactants and organic solvents. Theadditional kit components may be present as pure compositions, or asaqueous solutions that incorporate one or more additional kitcomponents. Any or all of the kit components optionally further comprisebuffers.

In one aspect of the disclosed kit, the kit includes at least oneindicator compound as described above, and a non-fluorescent andcell-impermeant quencher dye. The non-fluorescent and cell-impermeantquencher dye is optionally present in a combined buffer solution withthe compound, or the buffer solution of the cell-impermeant quencher dyeis present in a separate container from the indicator compound.

The examples provided below illustrate selected aspects of theinvention. They are not intended to limit or define the entire scope ofthe invention.

EXAMPLES Example 1 Preparation of Compounds 3 and 4

Compound 2 (20 g) is prepared according to the procedure of US PatentApplication US20120183986 (Diwu et al). To the solution of Bisphenol 1(5 g) in methanesulfonic acid (15 mL) Compound 2 is added with stirring.The resulting mixture is heated under dry nitrogen at 70-80° C. untilCompound 1 is mostly consumed. The cooled mixture is poured into icewater. The reaction mixture is stirred at room temperature overnight tocompletely convert partially demethylated Compounds 3 and 4 to themixture of free acid Compounds 3 and 4. The filtrate containing Compound3 and its isomer 4 are dried, and purified on a silica gel column elutedwith a gradient of water/acetonitrile to give the mixture of Compound 3and its isomer 4. The mixture of Compounds 3 and 4 is further purifiedby HPLC using C18 column and a gradient of 0.1% triethylammonium acetateacetonitrile-0.1% triethylammonium acetate buffer to give the pureCompounds 3 and 4.

Example 2 Preparation of Compound 8

Compound 3 (50 mg) is heated at 80° C. with Ac₂O (5 mL) and pyridine(0.1 mL) until Compound 3 is completely consumed. The solution is cooledto room temperature. The reaction mixture is poured into ice water, andcarefully adjusted to pH=4-5. The aqueous mixture is titrated withdioxane to give a precipitate that is collected by filtration. Theresulting mixture is first air-dried, and further vacuum-dried in adesiccator with P₂O₅ for 12 hours to yield crude Compound 7 that isdirectly used for next step reaction.

The crude Compound 7 (50 mg) is dissolved in anhydrous DMF (2 mL) atroom temperature. To the solution of Compound 7 BrCH₂OAc (0.1 mL) wasslowly added while stirring in a water bath. To the resulted mixtureiPr₂NEt (0.3 mL) is added slowly. The reaction mixture is stirred untilCompound 7 is completely consumed and concentrated in vacuo. The residueis suspended in ethyl acetate (20 mL) and stirred for 1-2 hours. Themixture is filtered to remove the solid that is washed with ethylacetate, and the filtrate is evaporated to dryness. The filtrate residueis purified on a silica gel column using 3:1:1 hexanes/EtOAc/chloroformas an eluent to give the desired Compound 8.

Example 3 Preparation of Compound 9

Compound 9 is prepared from Compound 4 analogously to the procedure ofCompound 8.

Example 4 Preparation of Compounds 11 and 12

The mixture of Compounds 11 and 12 are prepared from the reaction ofBisphenol 10 with Compound 2 analogously to the procedure of Compounds 3and 4. The mixed Compound 11 and its isomer 12 are further purified byHPLC using C18 column and a gradient of 0.1% triethylammonium acetateacetonitrile-0.1% triethylammonium acetate buffer to give the pureCompound 11 and 12.

Example 5 Preparation of Compound 16

Compound 16 is prepared from Compound 11 analogously to the procedure ofCompound 8.

Example 6 Preparation of Compound 18

Compound 18 is prepared from Compound 12 analogously to the procedure ofCompound 8.

Example 7 Preparation of Compound 20

Methyl 2-Bromo-4-hydroxybenzoate (100 g) is suspended in acetic acid(200 ml), and cooled to 0° C. To the suspension is dropwise added fumingnitric acid (45 ml). The reaction mixture is stirred at 0° C. for 2hours, and slowly warmed to room temperature. The reaction is continueduntil methyl 2-bromo-4-hydroxybenzoate is completely consumed. Thereaction mixture is poured to ice water, and the resulted mixture isfiltered to give yellow solid product that is further purified on asilica gel column using a gradient of methanol and chloroform as eluent.

Example 8 Preparation of Compound 25

Compound 25 is analogously prepared according to the procedure of U.S.Application No. 2002/0164616. A mixture of Compound 22 (15 g) and1-bromo-2-chloroethane (50 g) is dissolved in DMF at room temperature.To the reaction mixture K₂CO₃ is added with stirring. The reactionmixture is stirred at room temperature for 4-6 days. The reactionmixture is poured into water, and the resulted solid is collected. Thedried solid is purified on a silica gel column using a gradient ofhexanes/ethyl acetate to give a light yellow solid.

Example 9 Preparation of Compound 30

The mixture of Compound 20 (20 g) and 25 (20 g) is dissolved in DMF atroom temperature. To the reaction mixture K₂CO₃ is added, and thereaction mixture is stirred at 140-160° C. for 12-24 h. The reactionmixture is cooled, and poured into water, and resulted solid iscollected. The dried solid is purified on a silica gel column using agradient of hexanes/ethyl acetate to give a very light yellow solid.

Example 10 Preparation of Compound 35

Compound 30 (30 g) is dissolved in methanol (200 ml) at roomtemperature. To the MeOH solution is added stannous chloride dihydrate(25 g). The reaction mixture is heated with stirring at 70-80° C. untilCompound 30 is completely consumed. The reaction mixture is cooled toroom temperature, and poured into ice/water. The resulted suspension isextracted with ethyl acetate (3×500 ml), and combined organic phase isdried over anhydrous sodium sulfate. The solution is evaporated undervacuum to give a crude solid. The dried solid is purified on a silicagel column using a gradient of chloroform/ethyl acetate to give anoff-white solid.

Example 11 Preparation of Compound 45

To the solution of Compound 40 (20 g) and (iPr)₂NEt (50 mL) in DMF (100ml) is added tert-butyl bromoacetate (100 mL) with stirring. Thereaction mixture is refluxed until Compound 40 is completely consumed.The concentrated DMF solution is poured into water. The formed solid iscollected by filtration, and washed with water. The dried solid ispurified on a silica gel column using a gradient of chloroform/ethylacetate to give an off-white solid.

Example 12 Preparation of Compound 50

To the solution of Compound 45 (5 g) in methanol (100 ml) is added withstirring 1 M KOH (20 mL) is added with stirring at 0° C. The reactionmixture is stirred at 0° C. until the maximum amount of Compound 45monoacid is formed. The reaction mixture is carefully neutralized to pH7.5 with 1M HCl. The concentrated MeOH solution is poured into water.The formed solid is collected by filtration, and washed with water. Thecrude solid is dried under high vacuum to give an off-white solid. Thecompletely dried solid is dissolved in dichloromethane (100 ml), andcooled to 0° C. To the dichloromethane solution is added oxalyl chloride(5 ml) at 0° C., and followed with the addition of 3 drops of DMF. Thereaction mixture was warmed to room temperature, and stirred for 6hours. The reaction solution was concentrated under vacuum, and residuewas added to cold ether to give a suspension. The suspension is filteredto collect the formed solid that is completely dried under high vacuum.The dried solid is immediately dissolved in dichloromethane (50 ml). Tothe dichloromethane solution is dropwise added 2,2-dimethylethanolamine(5 ml), and followed by the dropwise addition of triethylamine (5 ml).The reaction solution is stirred at room temperature until Compound 45monoacyl chloride is consumed. The reaction solution is diluted withdichloromethane (150 ml), washed with saturated sodium bicarbonate andbrine. The organic phase is dried over anhydrous sodium sulfate, andevaporated under vacuum to give a gummy solid. The crude solid isdissolved in DMF (50 ml), and followed by the addition ofN,N′-dicyclohexylcarbodiimide (1 g). The DMF solution is stirred at −50°C. overnight, and filtered to collect the filtrate. The filtrate isconcentrated, and poured to water (100 ml) to collect the crude solid.The crude solid is dried and purified on a silica gel column using agradient of dichloromethane/ethyl acetate to give an off-white solid.

Example 13 Preparation of Compound 70

Compound 50 (10 g) is suspended in dichloromethane (40 ml). To thesuspension is added oxalyl chloride (8 ml), and followed by the additionof DMF (20 μL). The reaction mixture is stirred until Compound 50 iscompletely consumed. The reaction mixture is concentrated, followed byazeotrope with dichloromethane twice. The resulting oil is dried underhigh vacuum, and redissolved in dichloromethane (80 ml). To thedichloromethane solution is added Compound 51 (12 g), followed by theaddition of AlCl₃ (10.6 g) portionwise in ice bath. The reaction mixtureis stirred at room temperature until the amount of Compound 52 ismaximized, and the reaction mixture is diluted with dichloromethane andpoured onto ice. The mixture is sequentially washed with water andsaturated Na₂CO₃. The organic phase is dried by MgSO₄, filtered, andconcentrated to give the crude. The crude is purified on a silica gelcolumn with a gradient of hexanes/EtOAc to give Compound 52 as a whitesolid. Compound 52 is readily converted to Compound 56 according to theprocedure of J. B. Grimm et al. (ACS Chem Biol 2013, 8, 1303-1310).

Example 14 Preparation of Compound 79

Compound 79 is prepared analogously to the procedure of J. B Grimm etal. (ACS Chem Biol 2013, 8, 1303-1310). Compound 45 (1.2 g) is dissolvedin methyltetrahydrofuran, and the solution is cooled to −150° C. To thecold solution of Compound 45 is added 1.7 M t-BuLi (5 ml). The solutionis stirred at −150° C. for 2 hours. The solution of Compound 75 (1.1 g)in methyltetrahydrofuran (20 ml) is carefully added to maintain thereaction solution around −150° C. The reaction solution is stirred at−150° C. for 2 hours. To the reaction mixture is carefully added 1;1water/tetrahydrofuran (50 ml) to stop the reaction. The reaction mixtureis extracted with EtOAc, and the organic phase is washed with brine, anddried over sodium sulfate. The EtOAc solution is evaporated undervacuum, and the residue is redissolved in THF (50 ml). To the THFsolution is added tetrabutylammonium fluoride, and stirred until thesolution change to red. The solution is evaporated under vacuum, andredissolved in EtOAc. The EtOAc solution is washed with water for threetimes, and dried over sodium sulfate. The resulted EtOAC solution isevaporated under vacuum, and the residue is suspended in 20% HCl (50ml), and heated at 70-80° C. until the amount of Compound 79 ismaximized. The crude product is further purified on a C18 reverse phasesilica gel column with a gradient of triethylammonium bicarbonatebuffer/acetonitrile.

Example 15 Preparation of Compound 85

Compound 85 is prepared from Compound 79 analogously to the procedure ofCompound 8.

Example 16 Preparation of Compound 89

Compound 87 (200 mg) is dissolved in methyltetrahydrofuran, and solutionis cooled to −150° C. To the cold solution of Compound 87 is added 1.7 Mt-BuLi (1 ml). The solution is stirred at −150° C. for 1 hour. Thesolution of Compound 75 (180 mg) in methyltetrahydrofuran (5 ml) iscarefully added to maintain the reaction solution around −150° C. Thereaction solution is stirred at −150° C. for 1 hour. To the reactionmixture is carefully added 1:1 water/tetrahydrofuran (10 ml) to stop thereaction. The reaction mixture is extracted with EtOAc, and the organicphase is washed with brine, and dried over sodium sulfate. The EtOAcsolution is evaporated under vacuum, and the residue is redissolved inTHF (10 ml). To the THF solution is added tetrabutylammonium fluoride,and stirred until the solution change to red. The solution is evaporatedunder vacuum, and redissolved in EtOAc. The EtOAc solution is washedwith water for three times, and dried over sodium sulfate. The resultedEtOAC solution is evaporated under vacuum, and the residue is suspendedin 20% HCl (5 ml), and heated at 70-80° C. until the amount of Compound89 is maximized. The crude product is further purified on a C18 reversephase silica gel column with a gradient of triethylammonium bicarbonatebuffer/acetonitrile.

Example 17 Preparation of Compound 93

Compound 93 is prepared from Compound 89 analogously to the procedure ofCompound 8.

Example 18 Preparation of Compound 97

Compound 95 is prepared according to the procedure of J. B. Grimm et al.(ACS Chem Biol 2013, 8, 1303-1310). To the solution of Compound 95 (0.1g) in MeOH (5 ml) is dropwise added the mixture of NaOCl (0.6 ml, 10%aqueous solution) and 0.1 N NaOH (4 ml) at room temperature. Multipleportions of the mixed solution of NaOCl (0.4 ml) and 0.1 N NaOH (3 ml)is added at every hour until Compound 95 is completely consumed. Thereaction is neutralized with 0.2 N HCl. The mixture is extracted withEtOAc and the organic phase is dried with anhydrous Na₂SO₄, filtered,and concentrated to give the crude solid. The crude material is furtherpurified on a silica gel column with a gradient of hexanes/EtOAc to giveCompound 96 as a light yellow solid. To the solution of Compound 96(0.31 g) in THF (15 ml) is added iPr₂NEt (0.69 ml) and MOM-Cl (1.2 ml,2M toluene solution) in ice bath. The reaction is stirred at roomtemperature until Compound 96 is completely consumed. The reaction isworked with the addition of saturated aqueous NH₄Cl. The mixture isextracted with EtOAc and the organic phase is dried with anhydrousNa₂SO₄, filtered, and concentrated to give the crude solid. The crudematerial is further purified on a silica gel column with a gradient ofhexanes/EtOAc to give Compound 97 as a pale yellow wax.

Example 19 Preparation of Compound 100

2-Bromo-4-hydroxybenzoic acid (100 g) is suspended in acetic acid (200ml), and cooled to 0° C. To the suspension is dropwise added fumingnitric acid (45 ml) at 0° C. The reaction mixture is stirred at 0° C.for 2 hours, and slowly warmed to room temperature. The reaction iscontinued until 2-Bromo-4-hydroxybenzoic acid is mostly consumed. Thereaction mixture is poured to ice water, and the resulted mixture isfiltered to give yellow solid product that is further purified on asilica gel column using a gradient of methanol and chloroform as eluent.2-Bromo-4-hydroxy-5-nitrobenzoic acid is converted to Compound 100according the procedure of U. Schmidt et al. (J. Org. Chem. 1983, 48,2680-2685).

Example 20 Preparation of Compound 105

Compound 25 is analogously prepared according to the procedure of U.S.Pat. No. 4,689,432.

Example 21 Preparation of Compound 110

The mixture of Compound 105 (40 g) and 100 (20 g) is dissolved in DMF atroom temperature. To the reaction mixture K₂CO₃ is added, and thereaction mixture is stirred at 140-160° C. for 12-24 h. The reactionmixture is cooled, and poured into water, and resulted solid iscollected. The dried solid is purified on a silica gel column using agradient of hexanes/ethyl acetate to give a very light yellow solid.

Example 22 Preparation of Compound 113

Compound 110 is reduced to Compound 113 analogously to the procedure ofCompound 35.

Example 23 Preparation of Compound 115

Compound 113 is converted to Compound 115 by t-butylbromoacatealkylation analogously to the procedure of Compound 45.

Example 24 Preparation of Compound 120

Compound 115 (220 mg) is dissolved in methyltetrahydrofuran (3 ml), andsolution is cooled to −150° C. To the cold solution of Compound 115 isadded 1.7 M t-BuLi (0.9 ml). The solution is stirred at −150° C. for 1hour. To the solution of Compound 115 the solution of Compound 97 (240mg) in methyltetrahydrofuran (3 ml) is carefully added to maintain thereaction solution around −150° C. The reaction solution is stirred at−150° C. for 2 hours. To the reaction mixture is carefully added 1:1water/tetrahydrofuran (5 ml) to stop the reaction. The reaction mixtureis extracted with EtOAc, and the organic phase is washed with brine, anddried over sodium sulfate. The EtOAc solution is evaporated undervacuum, and the residue is redissolved in dichloromethane (3 ml). To thedichloromethane solution is added trifluoroacetic acid (3 ml), andfollowed by the addition of anisole (0.1 ml), and stirred at roomtemperature until the solution change to red. The solution is evaporatedunder vacuum, and redissolved in water. The aqueous solution is washedwith EtOAc for three times. The resulted aqueous solution isconcentrated under high vacuum, and the residue is further purified on aC18 reverse phase silica gel column with a gradient of triethylammoniumbicarbonate buffer/acetonitrile.

Example 25 Preparation of Compound 125

Compound 125 is prepared from Compound 122 analogously to the procedureof Compound 8. Compound 122 (40 mg) is heated at 80° C. with Ac₂O (5 mL)and pyridine (0.1 mL) until Compound 122 is completely consumed. Thesolution is cooled to room temperature. The reaction mixture is pouredinto ice water, and carefully adjusted to pH=4-5. The aqueous mixture istitrated with dioxane to give a precipitate that is collected byfiltration. The resulting mixture is first air-dried, and furthervacuum-dried in a desiccator with P₂O₅ for 12 hours to yield crudeCompound 123 that is directly used for next step reaction.

The crude Compound 123 (40 mg) is dissolved in anhydrous DMF (2 mL) atroom temperature. To the solution of Compound 123 BrCH₂OAc (0.1 mL) wasslowly added while stirring in a water bath. To the resulted mixtureiPr₂NEt (0.3 mL) is added slowly. The reaction mixture is stirred untilCompound 123 is completely consumed and concentrated in vacuo. Theresidue is suspended in ethyl acetate (20 mL) and stirred for 1-2 hours.The mixture is filtered to remove the solid that is washed with ethylacetate, and the filtrate is evaporated to dryness. The filtrate residueis purified on a silica gel column using 3:1:1 hexanes/EtOAc/chloroformas an eluent to give the desired Compound 125.

Example 26 Preparation of Compound 128

Compound 128 (100 mg) is dissolved in anhydrous DMF (3 mL) at RT. To thesolution BrCH₂OAc (0.5 mL) is slowly added while stirring in a waterbath. To the resulted mixture iPr₂NEt (0.38 mL) is added slowly. Thereaction mixture is stirred for 24-36 hours, and concentrated in vacuo.The oily residue is purified on a silica gel column using 3:1:1EtOAc/hexanes/chloroform as an eluent to give the desired Compound 128.

Example 27 Preparation of Compounds 132 and 133

The mixture of free acid Compounds 132 and 133 are prepared from thereaction of Bisphenol 10 with Compound 130 analogously to the procedureof S. Gaillard et al. (Org. Lett. 2007, 9, 2629-2632).

Example 28 Preparation of Compounds 135

Compound 135 is analogously prepared according to the procedure ofCompounds 3 and 4.

Example 29 Preparation of Compound 139

Compound 139 is prepared from Compound 135 analogously to the procedureof Compound 8.

Example 30 Preparation of Compounds 143

Compound 142 and 143 is analogously prepared from Compound 140 accordingto the procedure of Compounds 3 and 4.

Example 31 Preparation of Compound 145

Compound 145 is prepared from Compound 142 analogously to the procedureof Compound 8.

Example 32 Preparation of Compounds 148 and 149

Compounds 148 and 149 are analogously prepared from Compound 146according to the procedure of Compounds 3 and 4.

Example 33 Preparation of Compound 150

Compound 149 (50 mg) is heated at 80° C. with Ac₂O (5 mL) and pyridine(0.1 mL) until Compound 149 is completely consumed. The solution iscooled to room temperature. The reaction mixture is poured into icewater, and carefully adjusted to pH=6-7. The aqueous mixture is titratedwith dioxane to give a precipitate that is collected by filtration. Theresulting mixture is first air-dried, and further vacuum-dried in adesiccator with P₂O₅ for 12 hours to yield crude Compound 150 that ispurified on a silica gel column using a gradient of chloroform/methanolas an eluent to give the desired Compound 150.

Example 34 Calcium Responses of the Fluorescent Indicators MeasuredUsing a Microplate Reader Equipped with an Automated Liquid HandlingSystem

Calcium flux assays are preferred methods in drug discovery forscreening G protein coupled receptors (GPCR). The fluorescent indicatorsof the invention provide a homogeneous fluorescence-based assay fordetecting the intracellular calcium mobilization. Cells expressing aGPCR of interest that signals through calcium are pre-loaded with theindicator AM esters (such as Fluo-3 AM, Fluo-4 AM, Compounds 8, 16, 85,125 or 128) which can cross cell membrane. Once inside the cell, thelipophilic blocking groups are cleaved by non-specific cell esterase,resulting in a negatively charged fluorescein dye that is well-retainedin cells, and its fluorescence is greatly enhanced upon binding tocalcium. When the sample cells are stimulated with screening compounds,the receptor triggers a release of intracellular calcium, which thengreatly increases the fluorescence of the intracellular indicators. Thecombination of long wavelength fluorescence properties, highsensitivity, and often a >100 times increase in fluorescence uponbinding with calcium makes the disclosed indicators well-suited formeasurement of cellular calcium.

Specifically, CHO cells stably transfected with muscarinic receptor 1are plated at 60,000 cells per 100 μl per well in F12 with 5% FBS and 1%L-glutamine in a 96-well black wall/clear bottom Costar plate, incubatedin 5% CO₂, 37° C. incubator overnight. The growth medium is removed andthe cells are incubated with 100 μL/well of 1-8 μM Fluo-4 AM or Compound85 in Hanks and HEPES buffer with 0 mM or 2.5 mM probenecid for 1 hourat room temperature. ATP (50 μl/well) is added by NOVOstar (BMG LabTech)or FLIPR (Molecular Devices) to achieve the final indicatedconcentration. A representative dose response is shown in FIGS. 3 and 4.In the absence of probenecid Compound 85 demonstrates the unexpectedbetter fluorescence intensity enhancement upon calcium stimulation thanthat of Fluo-4 AM.

Example 35 Calcium Responses of the Fluorescent Indicators MeasuredUsing a Fluorescence Imaging Device

Calcium flux assays are preferred methods in drug discovery forscreening G protein coupled receptors (GPCR). The fluorescent indicatorsof the invention provide a homogeneous fluorescence-based assay fordetecting the intracellular calcium mobilization. Cells expressing aGPCR of interest that signals through calcium are pre-loaded with theindicator AM esters (such as Fluo-3 AM, Fluo-4 AM, Compounds 8, 16, 85,125 or 128) which can cross cell membrane. Once inside the cell, thelipophilic blocking groups are cleaved by non-specific cell esterase,resulting in a negatively charged fluorescein dye that is well-retainedin cells, and its fluorescence is greatly enhanced upon binding tocalcium. When the sample cells are stimulated with screening compounds,the receptor triggers a release of intracellular calcium, which thengreatly increases the fluorescence of the intracellular indicators. Thecombination of long wavelength fluorescence properties, highsensitivity, and often a >100 times increase in fluorescence uponbinding with calcium makes the disclosed indicators well-suited formeasurement of intracellular calcium.

Specifically, CHO cells stably transfected with muscarinic receptor 1are plated at 60,000 cells per 100 μl per well in F12 with 5% FBS and 1%L-glutamine in a 96-well black wall/clear bottom Costar plate, incubatedin 5% CO₂, 37° C. incubator overnight. The growth medium is removed andthe cells are incubated with 100 μL/well of 1-8 μM Fluo-4 AM or Compound85 in Hanks and HEPES buffer with 0 mM or 2.5 mM probenecid for 1 hourat room temperature. ATP (50 μl/well) is added by NOVOstar (BMG LabTech)or FLIPR (Molecular Devices) to achieve the final indicatedconcentration. A representative dose response is shown in FIGS. 11 and12. In the absence of probenecid Compound 85 demonstrates the unexpectedbetter fluorescence intensity enhancement upon calcium stimulation thanthat of Fluo-4 AM.

Although the present invention has been shown and described withreference to the foregoing operational principles and preferredembodiments, it will be apparent to those skilled in the art thatvarious changes in form and detail may be made without departing fromthe spirit and scope of the invention. The present invention is intendedto embrace all such alternatives, modifications and variances that fallwithin the scope of the appended claims.

1-13. (canceled)
 14. A compound having the formula:

wherein A is acetyl or acetoxymethyl; R¹-R¹² are independently hydrogen,halogen, carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido,nitro, nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl,boronic acid, aryl or heteroaryl; or alkyl, or alkoxy that is itselfoptionally substituted one or more times by halogen, amino, hydroxy,phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or heteroaryl; R²⁰and R²¹ are independently alkyl, carboxyalkyl, aryl or heteroaryl; oralkyl, or alkoxy that is itself optionally substituted one or more timesby halogen, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronicacid, aryl or heteroaryl; and Z is an acyloxymethyl having 3-10 carbons.15. The compound according to claim 14, wherein A is acetyl oracetoxymethyl; R¹-R¹² are independently hydrogen, alkyl, aryl,heteroaryl or halogen; R²⁰ and R²¹ are independently alkyl, alkoxy, arylor heteroaryl; and Z is acetoxymethyl.
 16. The compound according toclaim 14, wherein A is acetyl or acetoxymethyl; R¹-R¹² are independentlyhydrogen, alkyl, fluoro or chloro; R²⁰ and R²¹ are alkyl; and Z isacetoxymethyl.
 17. The compound according to claim 14, having theformula:

wherein A is acetyl or acetoxymethyl; R¹, R², R⁵ and R⁶ areindependently hydrogen, chloro or fluoro; and R¹⁰ is hydrogen, chloro,fluoro, nitro, methyl, ethyl, propyl, butyl, pentyl or hexyl. 18.-38.(canceled)
 39. A method of monitoring intracellular calcium using acompound of claim 14, comprising: a) contacting a sample comprising acell with the compound; b) incubating the sample for a time sufficientfor the compound to be loaded into the cell and an indicator compound tobe generated intracellularly; c) illuminating the sample at a wavelengththat generates a fluorescence response from the indicator compound; d)detecting a fluorescence response from the indicator compound.
 40. Themethod of claim 39, further comprising: stimulating the cell; monitoringchanges in the intensity of the fluorescence response from the indicatorcompound; and correlating the changes in fluorescence intensity withchanges in intracellular calcium levels.
 41. The method of claim 40,further comprising adding a cell-impermeant and non-fluorescent dye tothe sample.
 42. A kit for performing a calcium assay, comprising acompound of claim
 14. 43. The kit of claim 42, wherein thenon-fluorescent and cell-impermeant quencher dye is present in a mixedpowder or mixed solution with the compound, or the cell-impermeantquencher dye is provided in a separate container.
 44. The compoundaccording to claim 17, wherein R¹⁰ is hydrogen or methyl.
 45. Thecompound according to claim 44, wherein A is acetyl, R² and R⁵ arechloro, R¹ and R⁶ are hydrogen, and R¹⁰ is methyl.
 46. The compoundaccording to claim 44, wherein A is acetyl, R² and R⁵ are fluoro, R¹ andR⁶ are hydrogen, and R¹⁰ is methyl.
 47. The compound according to claim44, wherein A is acetyl, R² and R⁵ are chloro, R¹ and R⁶ are chloro, andR¹⁰ is hydrogen.
 48. The compound according to claim 44, wherein A isacetoxymethyl, R² and R⁵ are chloro, R¹ and R⁶ are hydrogen, and R¹⁰ ismethyl.